TAO protein kinases and methods of use therefor

ABSTRACT

Compositions and methods are provided for potentiating the activity of the mitogen-activated protein kinase p38. In particular the mitogen-activated protein kinase kinase MEK6, and variants thereof that stimulate phosphorylation of p38 are provided. Such compounds may be used, for example, for therapy of diseases associated with the p38 cascade and to identify antibodies and other agents that inhibit or activate signal transduction via p38.

This is a divisional of application Ser. No. 09/060,410 filed Apr. 14, 1998, now U.S. Pat. No. 6,165,461 issued Dec. 26, 2000, which is incorporated by reference herein in its entirety.

STATEMENT OF GOVERNMENT INTEREST

The Government owns certain rights in the present invention pursuant to NIH Grant GM53032.

TECHNICAL FIELD

The present invention relates generally to compositions and methods for modulating the activity of the MAP/ERK kinase MEK3 and/or other MEK family members. The invention is more particularly related to TAO proteins, and variants thereof that stimulate phosphorylation and activation of MEK substrates, such as MEK3. The invention is further related to the use of such proteins, for example, to activate a stress-responsive MAP kinase pathway in an organism and to identify antibodies and other agents that inhibit or activate signal transduction via such a pathway.

BACKGROUND OF THE INVENTION

MAP kinase pathways are conserved signal transduction pathways that activate transcription factors, translation factors and other target molecules in response to a variety of extracellular signals. Each pathway contains a MAP kinase module, consisting of a MAP kinase or ERK, a MAP/ERK kinase (MEK), and a MEK kinase (MEKK). In higher eucaryotes, activation of MAP kinase pathways has been correlated with cellular events such as proliferation, oncogenesis, development and differentiation. Accordingly, the ability to regulate signal transduction via these pathways could lead to the development of treatments and preventive therapies for human diseases associated with MAP kinase pathways, such as inflammatory diseases, autoimmune diseases and cancer.

Several MAP kinase pathways have been found in S. cerevisiae (Hunter and Plowman, Trends in Biochem. Sci. 22:18-22, 1997), and parallel mammalian pathways have been identified based upon sequences of mammalian ERKs and yeast MAP kinases, KSS1 and FUS3 (Boulton et al., Science 249:64-67, 1990; Courchesne et al., Cell 58: 1107-1119, 1989; Elion et al., Cell 60:649-664, 1990). The best delineated yeast MAP kinase pathway, activated by mating pheromones, is controlled by a receptor-G protein system, includes a Cdc42 small G protein, and requires at least three protein kinases, Ste20p (Leberer et al., EMBO J. 11:4815-4828, 1992; Ramer et al., Proc. Natl. Acad. Sci. USA 90:452-456, 1993), Ste11p (Rhodes et al., Genes Dev. 4:1862-1874, 1990), and Ste7p (Teague et al., i Proc. Natl. Acad Sci. USA 83:7371-7375, 1986), upstream of the MAP kinase Fus3p (Elion et al., Cell 60:649-664, 1990).

Ste20p was isolated from S. cerevisiae as a gene whose product functions downstream of the βγ subunits of a heterotrimeric G protein but upstream of enzymes in the MAP kinase module (MEKK, MEK, ERK) of the pheromone response pathway (Leberer et al., EMBO J. 11:4815-4828, 1992; Ramer et al., Proc. Natl. Acad. Sci. USA 90:452-456, 1993). Ste11p, the MEKK, may be one of the Ste20p substrates (Wu et al., J. Biol. Chem. 270:15984-15992, 1990); thus, Ste20-like enzymes may activate MEKKs in mammalian MAP kinase pathways. Ste20p, like its best studied mammalian counterparts, the p21-activated protein kinases (PAKs), is thought to be regulated by binding to Cdc42 through a conserved Cdc42/Rac interactive binding region, or CRIB domain (Burbelo et al., J. Biol. Chem. 270:29071-29074, 1995).

Mammalian relatives of Ste20p are diverse and include the PAK subfamily (PAK1,2,3) and the mixed lineage kinase (MLK) subfamily, including the dual leucine zipper kinase (DLK), germinal center kinase (GCK), and the Nck-interacting kinase, NIK. In the past year, newly identified Ste20p-related kinases include members of the MLK subfamily, SOK-1, Krs-1 and -2, and MUK. MUK was isolated in a screen for MEKK isoforms, but in fact shows more identity to MLK. In transfected cells several of these enzymes, as first shown with GCK, increase the activity of the stress-responsive kinases, particularly SAPK/JNK. In the case of NIK and GCK, they may work by binding to MEKK (Su et al., EMBO J. 16:1279-1290, 1997). However, several of these Ste20p-related enzymes also have MEKK activity. For example, DLK phosphorylates and potently activates MEKs that lie in the stress-responsive cascades.

Further characterization of members of these pathways, and the identification of additional members, is critical for understanding the signal transduction pathways involved and for developing methods for activating or inactivating MEKs and MAP kinase pathways in vivo. Accordingly, there is a need in the art for improved methods for modulating the activity of members of MAP kinase pathways, and for treating diseases associated with such pathways. The present invention fulfills these needs and further provides other related advantages.

SUMMARY OF THE INVENTION

Briefly stated, the present invention provides compositions and methods for modulating the activity of MAP/ERK kinases such as MEK3, and stress-responsive MAP kinase pathways. Within certain aspects, the present invention provides TAO polypeptides. Within one such aspect, the polypeptide may comprise an amino acid sequence provided in SEQ ID NO:2 or SEQ ID NO:4 or a variant thereof in which the ability to phosphorylate MEK3 is not substantially diminished. In certain embodiments, such a polypeptide may comprise a sequence that differs from a sequence recited in SEQ ID NO:2 or SEQ ID NO:4 only in conservative substitutions and/or modifications at no more than 10% of the amino acid residues. In certain other embodiments, the polypeptide may be a constitutively active variant.

Within other aspects, the present invention provides polypeptides comprising an amino acid sequence provided in SEQ ID NO:2 or SEQ ID NO:4 modified at no more than 10% of the amino acid residues, such that the polypeptide is rendered constitutively inactive.

Within further aspects, the present invention provides polypeptides capable of phosphorylating MEK3, wherein the polypeptide does not detectably phosphorylate MEK1 or MEK2.

The present invention further provides, within other aspects, isolated polynucleotides encoding polypeptides as described above. Isolated polynucleotides comprising one or more sequences recited in any one of SEQ ID NOs:5-16, or a variant thereof, wherein the polynucleotide encodes a polypeptide capable of phosphorylating MEK3, are also provided. Polypeptides encoded by such polynucleotides are further provided. Recombinant expression vectors comprising any of the above polynucleotides, and host cells transformed or transfected with such expression vectors, are provided within related aspects.

Within other aspects, the present invention provides antisense polynucleotides comprising at least 10 nucleotides complementary to a polynucleotide as described above.

Within further aspects, pharmaceutical compositions are provided, comprising: (a) a polypeptide or polynucleotide as described above; and (b) a physiologically acceptable carrier.

The present invention further provides, within other aspects, methods for phosphorylating a MEK3 polypeptide, comprising contacting a MEK3 polypeptide with a polypeptide according to claim as described above, thereby phosphorylating the MEK3 polypeptide.

Within further aspects, the present invention provides methods for activating a member of a stress-responsive MAP kinase pathway in an organism, comprising administering to an organism a polypeptide as described above, and thereby activating a member of a stress-responsive MAP kinase pathway.

Within other aspects, methods are provided for phosphorylating a MEK3 polypeptide comprising contacting a MEK3 polypeptide with a polypeptide as described above, and thereby phosphorylating the MEK3 polypeptide.

The present invention further provides methods for activating a member of a stress-responsive MAP kinase pathway in an organism, comprising administering to an organism a polypeptide as described above, and thereby activating a member of the stress-responsive MAP kinase pathway.

Within further aspects, the present invention provides methods for screening for an agent that modulates signal transduction via a stress-responsive MAP kinase pathway, comprising: (a) contacting a candidate agent with a polypeptide as described above; and (b) subsequently measuring the ability of the polypeptide to modulate the activity of a MEK3 polypeptide, and thereby evaluating the ability of the compound to modulate signal transduction via a stress-responsive MAP kinase pathway.

The present invention further provides, within other aspects, monoclonal antibodies and antigen-binding fragments thereof that specifically bind to a polypeptide as described above. Such monoclonal antibodies or fragments thereof may inhibit the phosphorylation of MEK3 by the polypeptide. Pharmaceutical compositions comprising: (a) an antibody or antigen-binding fragment thereof as described above; and (b) a physiologically acceptable carrier are also provided.

Within other aspects, the present invention provides methods for treating a patient afflicted with a disease associated with a stress-responsive MAP kinase pathway, comprising administering to a patient a compound that modulates the phosphorylation of MEK3. Within certain embodiments, the compound comprises a monoclonal antibody or antigen-binding fragment thereof or a nucleotide sequence. Within such methods, the compound may inhibit phosphorylation of MEK3 and the disease may be inflammation, an autoimmune disease, cancer or a degenerative disease. Alternatively, the compound may enhance the phosphorylation of MEK3 and the disease may be insulin-dependent diabetes or a neurodegenerative disease.

Within other aspects, methods are provided for determining the presence or absence of TAO kinase activity in a sample, comprising evaluating the ability of the sample to phosphorylate a MEK3 polypeptide, and thereby determining the presence or absence of TAO kinase activity in the sample.

Within related aspects, kits are provided for detecting TAO kinase activity in a sample, comprising a MEK3 polypeptide in combination with a suitable buffer.

These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B present the nucleotide and predicted amino acid sequence of a representative TAO1 kinase (SEQ ID NOs: 1 and 2).

FIG. 2 presents a comparison of the catalytic domains of TAO1 (residues 1-273 of SEQ ID NO:2), TAO2 (residues 1-273 of SEQ ID NO:4), STE20 (SEQ ID NO:17) and the C. elegans homolog (ceTAO) (SEQ ID NO:18). The catalytic domains were aligned by eye and the conserved amino acids bolded. The domains are indicated with roman numerals.

FIGS. 3A and 3B are Northern blots, showing TAO1 (FIG. 3A) and TAO2 (FIG. 3B) expression is various tissues. Various rat poly-A+ RNAs were probed, as indicated. Equal loading of RNA was verified by hybridizing the blot to an actin probe (not shown).

FIGS. 4A and 4B are Northern blots in which RNAs made from various human brain and spinal cord sections were hybridized to a TAO1-specific probe. Shown below each blot is the result of its hybridization to an actin probe. The lanes are as follows: 1, amygdala, 2, caudate nucleus, 3, corpus callosum, 4, hippocampus, 5, whole brain, 6, substantia nigra, 7, subthalamic nucleus, 8, thalamus, 9, cerebellum, 10, cerebral cortex, 11, medulla, 12, spinal cord, 13, occipital lobe, 14, frontal lobe, 15, temporal lobe, 16, putamen.

FIGS. 5A-5C are immunoblots. In FIG. 5A, human embryonic kidney 293 cells were transiently transfected with either vector or pCMV5TAO1(HA)₃, and 24 hours later lysates were immunoblotted with a monoclonal antibody directed against the HA epitope. TAO1 is indicated by the arrow. In FIG. 5B, the TAO1 proteins purified from Sf9 cells were immunoblotted with an antibody directed against the MRGS(H)₆ epitope. In FIG. 5C, 50 ng of (His)₆TAO1 was immunoblotted with polyclonal antisera P820 directed against a TAO1 peptide. An equal amount was blotted with the preimmune serum for P820.

FIG. 6 is an autoradiogram showing the results of a representative in vitro linked kinase assay to estimate MEK activation by TAO1. Either 50 ng (lanes 1 and 3) or 250 ng (lanes 2 and 4) of (His)₆TAO1(1-416) was incubated with 50 ng of (His)₆MEK3 for one hour at 30° in the presence of Mg/ATP, after which a portion of the each reaction was added to a second reaction containing (His)₆p38. After a one hour incubation, the reactions were subjected to SDS-PAGE and autoradiography.

FIG. 7 is an autoradiogram showing the results of a representative in vitro linked kinase assay to estimate MEK activation by TAO1. Only the second part of the linked assay is shown. The assay was identical to that described in FIG. 6, except that GSTMEK4 was substituted for MEK3, and both (His)₆p38 and GSTSAPKβ were used as MEK4 substrates.

FIG. 8 is an autoradiogram showing the results of a representative in vitro linked kinase assay to estimate MEK activation by TAO1. The assay was as described in FIGS. 6 and 7, but was performed with GSTMEK6 and (His)₆p38 as the MEK6 substrate.

FIG. 9 is a histogram comparing the fold activations of MEKs 1 through 6 by (His)₆TAO1(1-416).

FIG. 10 is an autoradiogram illustrating TAO1 activation of MEK3 in vivo. Human embryonic kidney 293 cells were transiently transfected with either vector alone, or pCMV5TAO1(HA)₃ and pCMV5mycMEK3, alone and in combination. Immunoprecipitates made with a monoclonal antibody directed against the myc epitope were subjected to in vitro kinase assays with (His)₆p38 as substrate. Myc-tagged MEK3 expression detected with a polyclonal anti-MEK3 antisera is shown below. In several separate experiments, MEK3 activity in the immunoprecipitates was increased 3 to 4 fold when coexpressed with TAO1.

FIG. 11 is an autoradiogram illustrating the copurification of TAO1 and endogenous MEK3 from Sf9 cells. Either 100 μg of Sf9 whole cell lysate, or 1 μg each of the recombinant TAO1 proteins purified from Sf9 cells was Western blotted with polyclonal antisera directed against MEK3 (top panel) or MEK4 (lower panel). An identical Western blot performed with an antisera against MEK6 did not detect MEK6 protein in either the Sf9 lysate or the TAO1 preparations.

FIG. 12 presents an alignment of a human retina cDNA EST (sbjct; SEQ ID NO:5) with nts. 2341-2754 of the rat TAO1 kinase sequence (query) provided in FIG. 1 (SEQ ID NO:1).

FIG. 13 presents an alignment of a human retina cDNA EST (sbjct; SEQ ID NO:6) with nts. 964-651 of the rat TAO1 kinase sequence (query) provided in FIG. 1 (SEQ ID NO:1).

FIG. 14 presents an alignment of a human retina cDNA EST (sbjct; SEQ ID NO:7) with nts. 2792-2423 of the rat TAO1 kinase sequence (query) provided in FIG. 1 (SEQ ID NO:1).

FIG. 15A presents an alignment of a human retina cDNA EST (sbjct; SEQ ID NO:8) with nts. 2248-2437 of the rat TAO1 kinase sequence (query) provided in FIG. 1 (SEQ ID NO:1). FIG. 15B presents an alignment of a human retina cDNA EST (sbjct; SEQ ID NO:9) with nts. 2437-2501 of the rat TAO1 kinase sequence (query) provided in FIG. 1 (SEQ ID NO:1).

FIG. 16 presents an alignment of a human retina cDNA EST (sbjct; SEQ ID NO:10) with nts. 2087-2305 of the rat TAO1 kinase sequence (query) provided in FIG. 1 (SEQ ID NO:1).

FIG. 17A presents an alignment of a human retina cDNA EST (sbjct; SEQ ID NO:11) with nts. 3228-3312 of the rat TAO1 kinase sequence (query) provided in FIG. 1 (SEQ ID NO:1). FIG. 17B presents an alignment of a human retina cDNA EST (sbjct; SEQ ID NO:12) with nts. 3200-3245 of the rat TAO1 kinase sequence (query) provided in FIG. 1 (SEQ ID NO:1).

FIG. 18 presents an alignment of a human retina cDNA EST (sbjct; SEQ ID NO:13) with nts. 739-854 of the rat TAO1 kinase sequence (query) provided in FIG. 1 (SEQ ID NO:1).

FIG. 19A presents an alignment of a human retina cDNA EST (sbjct; SEQ ID NO:14) with nts. 526-643 of the rat TAO1 kinase sequence (query) provided in FIG. 1 (SEQ ID NO:1). FIG. 19B presents an alignment of a human retina cDNA EST (sbjct; SEQ ID NO:15) with nts. 187-296 of the rat TAO1 kinase sequence (query) provided in FIG. 1 (SEQ ID NO:1).

FIG. 20 presents an alignment of a human retina cDNA EST (sbjct; SEQ ID NO:16) with nts. 866-733 of the rat TAO1 kinase sequence (query) provided in FIG. 1 (SEQ ID NO:1).

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention is generally directed to compounds and methods for modulating (i.e., stimulating or inhibiting) the activity of MAP/ERK family members such as the MAP/ERK kinase MEK3. Compounds that activate such MEKs generally stimulate MEK phosphorylation. Such compounds include Ste20p homologs referred to herein as TAO polypeptides (i.e., TAO1, TAO2 or a variant of TAO1 or TAO2 that retains the ability to stimulate MEK3 phosphorylation at a level that is not substantially lower than the level stimulated by the native protein). Alternatively, a compound that activates MEK3 may comprise a polynucleotide that encodes a TAO polypeptide. Within other embodiments, compositions that stimulate MEK3 phosphorylation (thereby activating MEK3) may also, or alternatively, include one or more agents that stimulate TAO polypeptide expression or kinase activity. Such agents include, but are not limited to, stress-inducing agents (e.g., DNA-damaging agents). Additional such agents may be identified by combining a test compound with a TAO polypeptide in vitro and evaluating the effect of the test compound on the kinase activity of the polypeptide using, for example, a representative assay described herein.

Compositions that inhibit the activity of MEKs generally inhibit MEK phosphorylation. Such compositions may include one or more agents that inhibit or block TAO polypeptide activity, such as an antibody that inhibits the kinase activity of a TAO polypeptide, a competing peptide that represents the substrate binding domain of a TAO protein or a phosphorylation motif of the MEK3 substrate, an antisense polynucleotide or ribozyme that interferes with transcription and/or translation of a TAO polypeptide, a molecule that inactivates a TAO polypeptide by binding to the polypeptide, a molecule that binds to the TAO substrate and prevents phosphorylation by a TAO polypeptide or a molecule that prevents transfer of phosphoryl groups from the kinase to the substrate. Agents that inhibit TAO polypeptide kinase activity may be identified by combining a test compound with a TAO polypeptide in vitro and evaluating the activity of the TAO polypeptide using a TAO kinase assay.

Tao Polynucleotides

Any polynucleotide that encodes a TAO polypeptide, or a portion or variant thereof as described herein, is encompassed by the present invention. Such polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide of the present invention, and a TAO polynucleotide may, but need not, be linked to other molecules and/or support materials.

Native TAO DNA sequences, or portions thereof, may be isolated using any of a variety of hybridization or amplification techniques, which are well known to those of ordinary skill in the art. Within such techniques, probes or primers may be designed based on the TAO sequences provided herein, and may be purchased or synthesized. Libraries from any suitable tissue (e.g., brain) may be screened. An amplified portion or partial cDNA molecule may then be used to isolate a full length gene from a genomic DNA library or from a cDNA library, using well known techniques. Alternatively, a full length gene can be constructed from multiple PCR fragments.

Nucleic acid sequences corresponding to the native rat TAO polypeptides TAO1 and TAO2 are provided in SEQ ID NO:1 and SEQ ID NO:3, respectively. One preferred variant of TAO1 comprises amino acids 1-416 of SEQ ID NO:1. The predicted TAO1 open reading frame encodes a polypeptide of 1001 amino acids with a calculated molecular mass of 134 kD. TAO1 comprises an amino-terminal catalytic domain and an extensive carboxy-terminal region that has several distinguishing features, such as a possible nucleotide binding site and acidic stretch just carboxy-terminal to the catalytic domain, as well as two serine-rich regions. The TAO1 catalytic domain extends 263 amino acids from amino acid 25 to 288 with all 11 of the typical protein kinase subdomains conserved. There are two glutamate residues between TAO1 subdomains II and IV; the second glutamate at amino acid 76 contained in the sequence KEVK is most likely to represent subdomain III (Hanks et al., Science 241:42-52, 1988). The features of the TAO1 catalytic domain are most similar to the serine/threonine family of protein kinases; subdomain VIb with the sequence HRDIKAGN (SEQ ID NO:26) suggests that TAO1 is likely to be a serine/threonine protein kinase.

A partial sequence of the closely related gene TAO2 is provided in SEQ ID NOs:3 and 4. TAO2 has a similar arrangement of an amino-terminal kinase domain and a long carboxy-terminus, but differs in that it contains an acidic insert of 17 glutamate residues carboxy-terminal to the catalytic domain, and lacks the putative nucleotide binding site of TAO1.

The rat TAO1 and TAO2 transcripts are highly expressed in brain. Lower levels of TAO1 expression can be observed in heart and lung, with no detectable signal (using a Northern blot analysis as described herein) in skeletal muscle, liver, kidney, testis, epididymus and spleen.

The polynucleotides specifically recited herein, as well as full length polynucleotides comprising such sequences, other portions of full length polynucleotides, and sequences complementary to all or a portion of such full length molecules, are specifically encompassed by the present invention. In addition, TAO homologs from other species are specifically contemplated, and may generally be prepared as described herein for the rat homologs. In particular, within the context of the present invention, EST database sequences derived from retinal mRNAs have been identified that correspond to the human counterpart for TAO1. The sequences of these ESTs are provided in SEQ ID NOs:5-16. It will be readily apparent to those of ordinary skill in the art that a full length, native, human TAO1 polynucleotide may be identified based on such sequences, using for example, standard hybridization or amplification techniques. Such full length TAO1 sequences are contemplated by the present invention, as are polypeptides encoded by such sequences, and variants of the naturally occurring sequences as discussed herein.

Polynucleotide variants of the recited sequences may differ from a native TAO polynucleotide in one or more substitutions, deletions, insertions and/or modifications. Certain variants encode a polypeptide that retains the ability to stimulate MEK3 phosphorylation at a level that is not substantially lower than the level stimulated by the native protein. The effect on the properties of the encoded polypeptide may generally be assessed as described herein. Preferred variants contain nucleotide substitutions, deletions, insertions and/or modifications at no more than 20%, preferably at no more than 10%, of the nucleotide positions. Certain variants are substantially homologous to a native gene, or a potion or complement thereof. Such polynucleotide variants are capable of hybridizing under moderately stringent conditions to a naturally occurring DNA sequence encoding a TAO protein (or a complementary sequence). Suitable moderately stringent conditions include prewashing in a solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50° C.-65° C., 5×SSC, overnight; followed by washing twice at 65° C. for 20 minutes with each of 2×, 0.5×and 0.2×SSC containing 0.1% SDS). Such hybridizing DNA sequences are also within the scope of this invention.

It will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide as described herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention.

As noted above, the present invention further provides antisense polynucleotides and portions of any of the above sequences. Such polynucleotides may generally be prepared by any method known in the art, including synthesis by, for example, solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro or in vivo transcription of DNA sequences that are incorporated into a vector downstream of a suitable RNA polymerase promoter (such as T3, T7 or SP6). Certain portions of a TAO polynucleotide may be used to prepare an encoded polypeptide, as described herein. In addition, or alternatively, a portion may function as a probe (e.g., to detect TAO expression in a sample), and may be labeled by a variety of reporter groups, such as radionuclides, fluorescent dyes and enzymes. Such portions are preferably at least 10 nucleotides in length, and more preferably at least 20 nucleotides in length. Within certain preferred embodiments, a portion for use as a probe comprises a sequence that is unique to a TAO gene. A portion of a sequence complementary to a coding sequence (i.e., an antisense polynucleotide) may also be used as a probe or to modulate gene expression. DNA constructs that can be transcribed into antisense RNA may also be introduced into cells or tissues to facilitate the production of antisense RNA.

Any polynucleotide may be further modified to increase stability in vivo. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5′ and/or 3′ ends; the use of phosphorothioate or 2′ O-methyl rather than phosphodiesterase linkages in the backbone; and/or the inclusion of nontraditional bases such as inosine, queosine and wybutosine, as well as acetyl- methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine and uridine.

Nucleotide sequences as described herein may be joined to a variety of other nucleotide sequences using established recombinant DNA techniques. For example, a polynucleotide may be cloned into any of a variety of cloning vectors, including plasmids, phagemids, lambda phage derivatives and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors and sequencing vectors. In general, a vector will contain an origin of replication functional in at least one organism, convenient restriction endonuclease sites and one or more selectable markers. Additional initial, terminal and/or intervening DNA sequences that, for example, facilitate construction of readily expressed vectors may also be present. Suitable vectors may be obtained commercially or assembled from the sequences described by methods well-known in the art. Other elements that may be present in a vector will depend upon the desired use, and will be apparent to those of ordinary skill in the art.

Vectors as described herein may generally be transfected into a suitable host cell, such as a mammalian cell, by methods well-known in the art Such methods include calcium phosphate precipitation, electroporation and microinjection.

Tao Polypeptides

Polypeptides within the scope of the present invention comprise at least a portion of a TAO protein (e.g., TAO1 or TAO2) or variant thereof, where the portion is immunologically and/or biologically active. Preferred variants retain the ability to stimulate MEK3 phosphorylation at a level that is not substantially lower than the level stimulated by the native protein. A polypeptide may further comprise additional sequences, which may or may not be derived from a native TAO protein. Such sequences may (but need not) possess immunogenic or antigenic properties and/or a biological activity.

A polypeptide “variant,” as used herein, is a polypeptide that differs from a native protein in substitutions, insertions, deletions and/or amino acid modifications, such that the immunogenic and/or biological properties of the native protein are not substantially diminished. A variant preferably retains at least 80% sequence identity to a native sequence, more preferably at least 90% identity, and even more preferably at least 95% identity. Within certain preferred embodiments, such variants contain alterations at no more than 10% of the amino acid residues in the native polypeptide, such that the ability of the variant to stimulate MEK3 phosphorylation is not substantially diminished. Guidance in determining which and how many amino acid residues may be substituted, inserted, deleted and/or modified without diminishing immunological and/or biological activity may be found using any of a variety of methods and computer programs known in the art. Properties of a variant may generally be evaluated by assaying the reactivity of the variant with, for example, antibodies as described herein and/or evaluating a biological property characteristic of the native protein.

A polypeptide is “immunologically active,” within the context of the present invention if it is recognized (i.e., specifically bound) by a B-cell and/or T-cell surface antigen receptor. Immunological activity may generally be assessed using well known techniques, such as those summarized in Paul, Fundamental Immunology, 3rd ed., 243-247 (Raven Press, 1993) and references cited therein. Such techniques include screening polypeptides derived from the native polypeptide for the ability to react with antigen-specific antisera and/or T-cell lines or clones, which may be prepared using well known techniques. An immunologically active portion of a TAO protein reacts with such antisera and/or T-cells at a level that is not substantially lower than the reactivity of the full length polypeptide (e.g., in an ELISA and/or T-cell reactivity assay). Such screens may generally be performed using methods well known to those of ordinary skill in the art, such as those described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. B-cell and T-cell epitopes may also be predicted via computer analysis.

Similarly, a polypeptide is “biologically active” if the ability to phosphorylate MEK3 and/or other MEKs is not substantially diminished within a representative in vitro assay as described in Example 3. Preferably, the ability of the polypeptide to phosphorylate MEK3 is not substantially diminished. As used herein, the term “not substantially diminished” means retaining an activity that is at least 90% of the activity of a native TAO protein. Appropriate assays designed to evaluate such activity may be designed based on existing assays known in the art, and on the representative assays provided herein.

Preferably, a variant contains conservative substitutions. A “conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. Amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may also, or alternatively, contain nonconservative changes.

In general, modifications may be more readily made in non-critical regions, which are regions of the native sequence that do not substantially change the properties of the TAO protein. Non-critical regions may be identified by modifying the TAO sequence in a particular region and assaying the activity of the resulting variant in a kinase assay, using MEK3, MEK4, MEK6 or another MEK family member as a substrate, as described herein. Modifications may also be made in critical regions of a TAO protein, provided that the resulting variant retains the ability to stimulate MEK3 phosphorylation and/or an immunogenic property of the native protein. Inactive proteins may be created by modifying certain critical regions. One critical region comprises the aspartate 169 residue. Modification of that residue results in a catalytically defective mutant. Another critical region encompasses the lysine 57 residue. The effect of any modification on the ability of the variant to stimulate phosphorylation of MEK3 or other MEKs may generally be evaluated using any assay for TAO kinase activity, such as the representative assays described herein.

Variants of TAO proteins include constitutively active proteins. In general, activation of a TAO protein in vivo requires stimulation by a stimulus such as a stress-inducing agent. Constitutively active variants display the ability to stimulate MEK phosphorylation in the absence of such stimulation. Such variants may be identified using the representative in vivo assays for TAO kinase activity described herein.

TAO proteins may also be modified so as to render the protein constitutively inactive (i.e., unable to phosphorylate MEKs even when stimulated as described above). Such modifications may be identified using the representative assays described herein. Genes encoding proteins modified so as to be constitutively active or inactive may generally be used in replacement therapy for treatment of a variety of disorders, as discussed in more detail below.

Variants within the scope of this invention also include polypeptides in which the primary amino acid structure of a native protein is modified by forming covalent or aggregative conjugates with other polypeptides or chemical moieties such as glycosyl groups, lipids, phosphate, acetyl groups and the like. Covalent derivatives may be prepared, for example, by linking particular functional groups to amino acid side chains or at the N- or C-termini.

The present invention also includes polypeptides with or without associated native-pattern glycosylation. Polypeptides expressed in yeast or mammalian expression systems may be similar to or slightly different in molecular weight and glycosylation pattern than the native molecules, depending upon the expression system. Expression of DNA in bacteria such as E. coli provides non-glycosylated molecules. N-glycosylation sites of eukaryotic proteins are characterized by the amino acid triplet Asn-A₁-Z, where A₁ is any amino acid except Pro, and Z is Ser or Thr. Variants having inactivated N-glycosylation sites can be produced by techniques known to those of ordinary skill in the art, such as oligonucleotide synthesis and ligation or site-specific mutagenesis techniques, and are within the scope of this invention. Alternatively, N-linked glycosylation sites can be added to a polypeptide.

As noted above, polypeptides may further comprise sequences that are not related to an endogenous TAO protein. For example, an N-terminal signal (or leader) sequence may be present, which co-translationally or post-translationally directs transfer of the polypeptide from its site of synthesis to a site inside or outside of the cell membrane or wall (e.g., the yeast α-factor leader). The polypeptide may also comprise a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His, hemagglutinin, glutathione-S-transferase or FLAG), or to enhance polypeptide stability or binding to a solid support. Protein fusions encompassed by this invention further include, for example, polypeptides conjugated to an immunoglobulin Fc region or a leucine zipper domain. All of the above protein fusions may be prepared by chemical linkage or as fusion proteins.

Also included within the present invention are alleles of a TAO protein. Alleles are alternative forms of a native protein resulting from one or more genetic mutations (which may be amino acid deletions, additions and/or substitutions), resulting in an altered mRNA. Allelic proteins may differ in sequence, but overall structure and function are substantially similar.

TAO polypeptides, variants and portions thereof may generally be prepared from nucleic acid encoding the desired polypeptide using well known techniques. To prepare an endogenous protein, an isolated cDNA may be used. To prepare a variant polypeptide, standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis may be used, and sections of the DNA sequence may be removed to permit preparation of truncated polypeptides.

In general, any of a variety of expression vectors known to those of ordinary skill in the art may be employed to express recombinant polypeptides of this invention. Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a DNA sequence that encodes a recombinant polypeptide. Suitable host cells include prokaryotes, yeast, baculovirus-infected insect cells and animal cells. Following expression, supernatants from host/vector systems which secrete recombinant protein or polypeptide into culture media may be first concentrated using a commercially available filter. Following concentration, the concentrate may be applied to a suitable purification matrix such as an affinity matrix or an ion exchange resin. One or more reverse phase HPLC steps can be employed to further purify a recombinant polypeptide.

Portions and other variants having fewer than about 100 amino acids, and generally fewer than about 50 amino acids, may also be generated by synthetic means, using techniques well known to those of ordinary skill in the art. For example, such polypeptides may be synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. See Merrifield, J. Am. Chem. Soc. 85:2149-2146, 1963. Various modified solid phase techniques are also available (e.g., the method of Roberge et al., Science 269:202-204, 1995). Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Applied BioSystems, Inc. (Foster City, Calif.), and may be operated according to the manufacturer's instructions.

In general, polypeptides and polynucleotides as described herein are isolated. An “isolated” polypeptide or polynucleotide is one that is removed from its original environment. For example, a naturally-occurring protein is isolated if it is separated from some or all of the coexisting materials in the natural system. Preferably, polypeptides provided herein are isolated to a purity of at least 80% by weight, more preferably to a purity of at least 95% by weight, and most preferably to a purity of at least 99% by weight. In general, such purification may be achieved using, for example, the standard techniques of ammonium sulfate fractionation, SDS-PAGE electrophoresis, and affinity chromatography. A polynucleotide is considered to be isolated if, for example, it is cloned into a vector that is not a part of the natural environment.

Antibodies and Fragments Thereof

The present invention further provides antibodies, and antigen-binding fragments thereof, that specifically bind to a TAO polypeptide. As used herein, an antibody, or antigen-binding fragment, is said to “specifically bind” to a TAO polypeptide if it reacts at a detectable level (within, for example, an ELISA) with a TAO polypeptide, and does not react detectably with unrelated proteins. Antibodies may be polyclonal or monoclonal. Preferred antibodies are those antibodies that inhibit or block TAO activity in vivo and within a kinase assay as described herein. Other preferred antibodies (which may be used, for example, in immunokinase assays) are those that immunoprecipitate active TAO1 and/or TAO2.

Antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). In one such technique, an immunogen comprising the polypeptide is initially injected into a suitable animal (e.g., mice, rats, rabbits, sheep and goats), preferably according to a predetermined schedule incorporating one or more booster immunizations, and the animals are bled periodically. Polyclonal antibodies specific for the polypeptide may then be purified from such antisera by, for example, affinity chromatography using the polypeptide coupled to a suitable solid support.

Monoclonal antibodies specific for a TAO polypeptide may be prepared, for example, using the technique of Kohler and Milstein, Eur. J. Immunol. 6:511-519, 1976, and improvements thereto. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity (i.e., reactivity with the polypeptide of interest). Such cell lines may be produced, for example, from spleen cells obtained from an animal immunized as described above. The spleen cells are then immortalized by, for example, fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal. For example, the spleen cells and myeloma cells may be combined with a nonionic detergent for a few minutes and then plated at low density on a selective medium that supports the growth of hybrid cells, but not myeloma cells. A preferred selection technique uses HAT (hypoxanthine, aminopterin, thymidine) selection. After a sufficient time, usually about 1 to 2 weeks, colonies of hybrids are observed. Single colonies are selected and tested for binding activity against the polypeptide. Hybridomas having high reactivity and specificity are preferred.

Monoclonal antibodies may be isolated from the supernatants of growing hybridoma colonies. In addition, various techniques may be employed to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. Monoclonal antibodies may then be harvested from the ascites fluid or the blood. Contaminants may be removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction.

Within certain embodiments, the use of antigen-binding fragments of antibodies may be preferred. Such fragments include Fab fragments, which may be prepared using standard techniques. Briefly, immunoglobulins may be purified from rabbit serum by affinity chromatography on Protein A bead columns (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988) and digested by papain to yield Fab and Fc fragments. The Fab and Fc fragments may be separated by, for example, affinity chromatography on protein A bead columns.

Methods and Kits for Detecting Tao Polypeptides and Tao Kinase Activity

The present invention provides methods for detecting the level of TAO1 and/or TAO2 in a sample, as well as for detecting TAO kinase activity in a sample. The level of a TAO polypeptide or polynucleotide may generally be determined using a reagent that binds to the TAO protein, DNA or mRNA. To detect nucleic acid encoding a TAO protein, standard hybridization and/or PCR techniques may be employed using a nucleic acid probe or a PCR primer. Suitable probes and primers may be designed by those of ordinary skill in the art based on the TAO cDNA sequences provided herein. To detect TAO protein, the reagent is typically an antibody, which may be prepared as described herein.

There are a variety of assay formats known to those of ordinary skill in the art for using an antibody to detect a polypeptide in a sample. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. For example, the antibody may be immobilized on a solid support such that it can bind to and remove the polypeptide from the sample. The bound polypeptide may then be detected using a second antibody that binds to the antibody/peptide complex and contains a detectable reporter group. Alternatively, a competitive assay may be utilized, in which polypeptide that binds to the immobilized antibody is labeled with a reporter group and allowed to bind to the immobilized antibody after incubation of the antibody with the sample. The extent to which components of the sample inhibit the binding of the labeled polypeptide to the antibody is indicative of the level of polypeptide within the sample. Suitable reporter groups for use in these methods include, but are not limited to, enzymes (e.g., horseradish peroxidase), substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups and biotin.

For detecting an active TAO protein in a sample, an immunokinase assay may be employed. Briefly, polyclonal or monoclonal antibodies may be raised against a unique sequence of a TAO protein (such as amino acid residues 296-315, 403-418, 545-563 or 829-848) using standard techniques. A sample to be tested, such as a cellular extract, is incubated with the anti-TAO antibodies to immunoprecipitate a TAO protein, and the immunoprecipitated material is then incubated with a substrate (e.g., MEK3) under suitable conditions for substrate phosphorylation. The level of substrate phosphorylation may generally be determined using any of a variety of assays, as described herein.

TAO kinase assays, for use in evaluating the polypeptide variants and other agents discussed herein, include any assays that evaluate a compound's ability to phosphorylate MEK3 or other MEKs, thereby rendering the MEK active (i.e., capable of phosphorylating in vivo substrates such as p38). MEKs such as MEK3 for use in such methods may be endogenous proteins or variants thereof, may be purified or recombinant, and may be prepared using any of a variety of techniques that will be apparent to those of ordinary skill in the art. For example, cDNA encoding MEK3 may be cloned by PCR amplification from a suitable human cDNA library, using polymerase chain reaction (PCR) and methods well known to those of ordinary skill in the art. MEK3 may be cloned using primers based on the published sequence (Derijard et al., Science 267:682-685, 1995). MEK3 cDNA may then be cloned into a bacterial expression vector and the protein produced in bacteria, such as E. coli, using standard techniques. The bacterial expression vector may, but need not, include DNA encoding an epitope such as glutathione-S transferase protein (GST) such that the recombinant protein contains the epitope at the N- or C-terminus.

A TAO kinase assay may generally be performed as described herein. Briefly, a TAO polypeptide may be incubated with MEK3 and [γ-³²P]ATP in a suitable buffer (such as 50 mM HEPES pH 8, 10 mM MgCl₂, 1 mM DTT, 100 μM ATP) for 60 minutes at 30° C. In general, approximately 50 ng to 1 μg of the polypeptide and 50 ng recombinant MEK3, with 2-7 cpm/fmol [γ-³²P]ATP, is sufficient. Proteins may then be separated by SDS-PAGE on 10% gels and subjected to autoradiography. Incorporation of [³²P]phosphate into MEK3 may be quantitated using techniques well known to those of ordinary skill in the art, such as with a phosphorimager. To evaluate the substrate specificity of polypeptide variants, a kinase assay may generally be performed as described above except that other MEK substrates (i.e., MEK1, 2, 4 or 6) are substituted for the MEK3.

To determine whether MEK3 phosphorylation results in activation, a coupled in vitro kinase assay may be performed using a substrate for MEK3, such as p38, with or without an epitope tag. p38 for use in such an assay may be prepared as described in Han et al., J. Biol Chem. 271:2886-2891, 1996. Briefly, following phosphorylation of MEK3 as described above, the MEK3 (e.g., 0.1-10 ng) may be incubated with p38 (e.g., 10 μg/ml) and [γ-³²P]ATP in a kinase buffer as described herein. It should be noted that alternative buffers may be used and that buffer composition can vary without significantly altering kinase activity. Reactions may be separated by SDS-PAGE, visualized by autoradiography and quantitated using any of a variety of known techniques. Activated MEK3 will be capable of phosphorylating p38 at a level that is at least 5% above background using such an assay.

The present invention further provides kits for detecting TAO polypeptides and TAO kinase activity. Such kits may be designed for detecting the level of a TAO polypeptide or polynucleotide, or may detect phosphorylation of MEK3 in a direct kinase assay or a coupled kinase assay, in which the level of phosphorylation and/or the kinase activity of MEK3 may be determined. TAO polypeptides and TAO kinase activity may be detected in any of a variety of samples, such as eukaryotic cells, bacteria, viruses, extracts prepared from such organisms and fluids found within living organisms. In general, the kits of the present invention comprise one or more containers enclosing elements, such as reagents or buffers, to be used in the assay.

A kit for detecting the level of TAO polypeptide or polynucleotide typically contains a reagent that binds to TAO1 and/or TAO2 protein, DNA or RNA. To detect nucleic acid encoding a TAO polypeptide, the reagent may be a nucleic acid probe or a PCR primer. To detect a TAO protein, the reagent is typically an antibody. The kit also contains a reporter group suitable for direct or indirect detection of the reagent (i.e., the reporter group may be covalently bound to the reagent or may be bound to a second molecule, such as Protein A, Protein G, immunoglobulin or lectin, which is itself capable of binding to the reagent). Suitable reporter groups include, but are not limited to, enzymes (e.g., horseradish peroxidase), substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups and biotin. Such reporter groups may be used to directly or indirectly detect binding of the reagent to a sample component using standard methods known to those of ordinary skill in the art.

A kit for detecting TAO kinase activity based on measuring the phosphorylation of MEK3 generally comprises MEK3 in combination with a suitable buffer. A kit for detecting TAO kinase activity based on detecting MEK3 activity generally comprises MEK3 in combination with a suitable MEK3 substrate, such as p38. Optionally, the kit may additionally comprise a suitable buffer and/or material for purification of MEK3 after activation and before combination with substrate. Such kits may be employed in direct or coupled kinase assays, which may be performed as described above.

Methods for Identifying Binding Agents and Modulating Agents

The present invention further provides methods for identifying antibodies and other compounds that bind to and/or modulate the activity of a TAO polypeptide. To evaluate the effect of a candidate modulating agent on TAO polypeptide activity, a kinase assay may be performed as described above, except that the candidate modulating agent is added to the incubation mixture. Briefly, the reaction components, which include the composition to be tested and the TAO polypeptide or a polynucleotide encoding the kinase, are incubated under conditions sufficient to allow the components to interact. Subsequently, the effect of composition on kinase activity or on the level of a polynucleotide encoding the kinase is measured. The observed effect on the kinase may be either inhibitory or stimulatory. The increase or decrease in kinase activity can be measured by, for example, adding a radioactive compound such as ³²P-ATP to the mixture of components, and observing radioactive incorporation into MEK3 or other suitable substrate for a TAO polypeptide, to determine whether the compound inhibits or stimulates kinase activity. A polynucleotide encoding the kinase may be inserted into an expression vector and the effect of a composition on transcription of TAO mRNA can be measured, for example, by Northern blot analysis.

Within such assays, the candidate agent may be preincubated with a TAO polypeptide before addition of ATP and substrate. Alternatively, the substrate may be preincubated with the candidate agent before the addition of kinase. Further variations include adding the candidate agent to a mixture of TAO polypeptide and ATP before the addition of substrate, or to a mixture of substrate and ATP before the addition of TAO polypeptide. Any of these assays can further be modified by removing the candidate agent after the initial preincubation step. In general, a suitable amount of antibody or other candidate agent for use in such an assay ranges from about 0.1 μM to about 10 μM. The effect of the agent on TAO kinase activity may then be evaluated by quantitating the incorporation of [³²P]phosphate into MEK3, as described above, and comparing the level of incorporation with that achieved using the TAO polypeptide without the addition of the candidate agent.

TAO kinase activity may also be measured in whole cells transfected with a reporter gene whose expression is dependent upon the activation of MEK3. For example, polynucleotides encoding a TAO polypeptide and a substrate (e.g., MEK3) may be cotransfected into a cell. The substrate may then be immunoprecipitated, and its activity evaluated in an in vitro assay. Alternatively, cells may be transfected with a ATF2-dependent promoter linked to a reporter gene such as luciferase. In such a system, expression of the luciferase gene (which may be readily detected using methods well known to those of ordinary skill in the art) depends upon activation of ATF2 by p38, which may be achieved by the stimulation of MEK3 with a TAO polypeptide. Candidate modulating agents may be added to the system, as described below. to evaluate their effect on TAO polypeptide activity.

Alternatively, a whole cell system may employ only the transactivation domain of ATF2 fused to a suitable DNA binding domain, such as GHF-1 or GAL4. The reporter system may then comprise the GH-luciferase or GAL4-luciferase plasmid. Candidate TAO protein modulating agents may then be added to the system to evaluate their effect on ATF2-specific gene activation.

In other aspects of the subject invention, methods for using the above polypeptides to phosphorylate and activate MEK3, peptide derivatives thereof or other MEK family members are provided. MEK substrate for use in such methods may be prepared as described above. In one embodiment, MEK3 may be phosphorylated in vitro by incubation with a TAO polypeptide and ATP in a suitable buffer as described above. In general, the amounts of the reaction components may range from about 0.1 μg to about 10 μg of TAO polypeptide, from about 0.1 μg to about 10 μg of recombinant MEK3, and from about 100 nM to about 1 mM (preferably about 100 pmol−30 nmol) of ATP. Phosphorylated proteins may then be purified by binding to GSH-sepharose and washing. The extent of MEK3 phosphorylation may generally be monitored by adding [γ-³²P]ATP to a test aliquot, and evaluating the level of MEK3 phosphorylation as described above. The activity of the phosphorylated MEK3 may be evaluated using a coupled in vitro kinase assay, as described above.

Once activated in vitro, MEK3 may be used, for example, to identify agents that inhibit the kinase activity of MEK3. Such inhibitory agents, which may be antibodies or drugs, may be identified using the coupled assay described above. Briefly, a candidate agent may be included in the mixture of MEK3 and p38, with or without pre-incubation with one or more components of the mixture, as described above. In general, a suitable amount of antibody or other agent for use in such an assay ranges from about 0.1 μM to about 10 μM. The effect of the agent on MEK3 kinase activity may then be evaluated by quantitating the incorporation of [³²P]phosphate into p38, as described above, and comparing the level of incorporation with that achieved using activated MEK3 without the addition of a candidate agent.

Within other aspects, TAO polypeptides may be used to identify one or more native upstream kinases (i.e., kinases that phosphorylate and activate TAO1 and/or TAO2 in vivo, or other signaling molecules that regulate TAO activity). TAO polypeptides may be used in a yeast two-hybrid system to identify interacting proteins. Alternatively, an expression library may be screened to identify cDNAs that encode proteins which phosphorylate a TAO polypeptide. Other methods for identifying such upstream kinases may also be employed, and will be apparent to those of ordinary skill in the art.

Pharmaceutical Compositions

For administration to a patient, one or more polypeptides, polynucleotides, antibodies and/or modulating agents are generally formulated as a pharmaceutical composition, which may be a sterile aqueous or non-aqueous solution, suspension or emulsion, and which additionally comprises a physiologically acceptable carrier (i.e., a non-toxic material that does not interfere with the activity of the active ingredient). Any suitable carrier known to those of ordinary skill in the art may be employed in a pharmaceutical composition. Representative carriers include physiological saline solutions, gelatin. water, alcohols, natural or synthetic oils, saccharide solutions, glycols, injectable organic esters such as ethyl oleate or a combination of such materials. Such compositions may also comprise buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, antimicrobial compounds, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), inert gases and/or preservatives. Compositions of the present invention may also be formulated as a lyophilizate. Pharmaceutical compositions may also contain other compounds, which may be biologically active or inactive.

The compositions described herein may be administered as part of a sustained release formulation (i.e., a formulation such as a capsule that effects a slow release of compound following administration). Such formulations may generally be prepared using well known technology and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site. Sustained-release formulations may contain a polypeptide, polynucleotide or modulating agent dispersed in a carrier matrix and/or contained within a reservoir surrounded by a rate controlling membrane. Carriers for use within such formulations are biocompatible, and may also be biodegradable; preferably the formulation provides a relatively constant level of release. The amount of active compound contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.

Certain pharmaceutical compositions contain DNA encoding a polypeptide, antibody fragment or other modulating agent as described above (such that a TAO polypeptide, a variant thereof or a modulating agent is generated in situ) or an antisense polynucleotide. In such pharmaceutical compositions, the DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid, bacterial and viral expression systems, as well as colloidal dispersion systems, including liposomes. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and terminating signal). The DNA may also be “naked,” as described, for example, in Ulmer et al., Science 259:1745-1749, 1993.

Various viral vectors that can be used to introduce a nucleic acid sequence into the targeted patient's cells include, but are not limited to, vaccinia or other pox virus, herpes virus, retrovirus, or adenovirus. Techniques for incorporating DNA into such vectors are well known to those of ordinary skill in the art. Preferably, the retroviral vector is a derivative of a murine or avian retrovirus including, but not limited to, Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). A retroviral vector may additionally transfer or incorporate a gene for a selectable marker (to aid in the identification or selection of transduced cells) and/or a gene that encodes the ligand for a receptor on a specific target cell (to render the vector target specific). For example, retroviral vectors can be made target specific by inserting a nucleotide sequence encoding a sugar, a glycolipid, or a protein. Targeting may also be accomplished using an antibody, by methods known to those of ordinary skill in the art.

Viral vectors are typically non-pathogenic (defective), replication competent viruses, which require assistance in order to produce infectious vector particles. This assistance can be provided, for example. by using helper cell lines that contain plasmids that encode all of the structural genes of the retrovirus under the control of regulatory sequences within the LTR, but that are missing a nucleotide sequence which enables the packaging mechanism to recognize an RNA transcript for encapsulation. Such helper cell lines include (but are not limited to) Ψ2, PA317 and PA12. A retroviral vector introduced into such cells can be packaged and vector virion produced. The vector virions produced by this method can then be used to infect a tissue cell line, such as NIH 3T3 cells, to produce large quantities of chimeric retroviral virions.

Another targeted delivery system for TAO polynucleotides is a colloidal dispersion system. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. A preferred colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (i.e., an artificial membrane vesicle). It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2-4.0 μm can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules. RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, et al., Trends Biochem. Sci. 6:77, 1981). In addition to mammalian cells, liposomes have been used for delivery of polynucleotides in plant, yeast and bacterial cells. In order for a liposome to be an efficient gene transfer vehicle, the following characteristics should be present: (1) encapsulation of the genes of interest at high efficiency while not compromising their biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Mannino, et al., Biotechniques 6:882, 1988).

The targeting of liposomes can be classified based on anatomical and mechanistic factors. Anatomical classification is based on the level of selectivity, for example, organ-specific, cell-specific, and organelle-specific. Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticuloendothelial system (RES) in organs which contain sinusoidal capillaries. Active targeting, on the other hand, involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.

Routes and frequency of administration, as well as polypeptide, modulating agent or nucleic acid doses, will vary from patient to patient. In general, the pharmaceutical compositions may be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity or transdermally. Between 1 and 6 doses may be administered daily. A suitable dose is an amount of polypeptide or DNA that is sufficient to show improvement in the symptoms of a patient afflicted with a disease associated with a stress-responsive MAP kinase pathway. Such improvement may be detected based on a determination of relevant cytokine levels (e.g., IL-2, IL-8), by monitoring inflammatory responses (e.g., edema, transplant rejection, hypersensitivity) or through an improvement in clinical symptoms associated with the disease. In general, the amount of polypeptide present in a dose, or produced in situ by DNA present in a dose, ranges from about 1 μg to about 250 μg per kg of host, typically from about 1 μg to about 60 μg. Suitable dose sizes will vary with the size of the patient, but will typically range from about 10 mL to about 500 mL for 10-60 kg animal.

Therapeutic Applications

The above polypeptides, polynucleotides and/or modulating agents may be used to phosphorylate (and thereby activate) MEK3, or to inhibit such phosphorylation, in a patient. As used herein, a “patient” may be any mammal, including a human, and may be afflicted with a disease associated with a stress-responsive MAP kinase pathway, or may be free of detectable disease. Accordingly, the treatment may be of an existing disease or may be prophylactic. Diseases associated with a stress-responsive MAP kinase pathway include any disorder which is etiologically linked to a TAO protein kinase activity, including immune-related diseases (e.g., inflammatory diseases, autoimmune diseases, malignant cytokine production or endotoxic shock), cell growth-related diseases (e.g., cancer, metabolic diseases, abnormal cell growth and proliferation or cell cycle abnormalities) and cell regeneration-related diseases (e.g., cancer, degenerative diseases, trauma, environmental stress by heat, UV or chemicals or abnormalities in development and differentiation). Immunological-related cell proliferative diseases such as osteoarthritis, ischemia, reperfusion injury, trauma, certain cancers and viral disorders, and autoimmune diseases such as rheumatoid arthritis, diabetes, multiple sclerosis, psoriasis, inflammatory bowel disease, and other acute phase responses may also be treated.

Treatment includes administration of a composition or compound which modulates the kinase activity of TAO1 and/or TAO2. Such modulation includes the suppression of TAO expression and/or activity when it is over-expressed, or augmentation of TAO expression and/or activity when it is under-expressed. Modulation may also include the suppression of phosphorylation of MEK3 or related kinases.

As noted above, antibodies, polynucleotides and other agents having a desired effect on TAO expression and/or activity may be administered to a patient (either prophylactically or for treatment of an existing disease) to modulate the activation of MEK3 in vivo. For example, an agent that decreases TAO activity in vivo may be administered to prevent or treat inflammation, autoimmune diseases, cancer or degenerative diseases. In particular, such agents may be used to prevent or treat insulin-resistant diabetes, metabolic disorders and neurodegenerative diseases. In general, for administration to a patient, an antibody or other agent is formulated as a pharmaceutical composition as described above. A suitable dose of such an agent is an amount sufficient to show benefit in the patient based on the criteria noted above.

The following Examples are offered by way of illustration and not by way of limitation.

EXAMPLES Example 1 Cloning and Sequencing cDNA Encoding TAO1 and TAO2

This Example illustrates the cloning of cDNA molecules encoding the at Ste20p-related protein kinases TAO1 and TAO2, and the identification of the human TAO1 homolog.

First-strand cDNA from adult rat brain was used as the template in the first round of PCR with degenerate oligonucleotide primers derived from the Ste20p sequence, 5′-GACGCTGGATCCAA(AG)AT(ACT)GGICA(AG)GGIGC-3′ (SEQ ID NO:19) and 5′-GGIGTICC(AG)TTIGTIGCIAT-3′ (SEQ ID NO:20). A portion of the product of this reaction was used as the template in a second round of PCR with nested primers, also derived from the Ste20p sequence, 5′-AA(AG)GA(AG)CAIATI(CA)TIAA(CT)GA(AG)AT-3′ (SEQ ID NO:21) and 5′-GACGCTGAATTCAC(CT)TCIGGIGCCATCCA-3′ (SEQ ID NO:22). The resulting 420 base product was labeled with [α⁻³²P]dCTP by random-priming, and used to probe approximately 1×10⁶ plaques of an oligo(dT) and random-primed λZAP library generated from adult rat forebrain RNA. In excess of 100 positive clones were obtained; of those sequenced, all contained regions of overlap with the original PCR product. A full length TAO1 sequence was assembled from two overlapping cDNAs, using the SacI site at nucleotide 50 to insert a fragment of TAO1 cDNA including nucleotides 50 to 3003. The full length TAO1 sequence is shown in FIG. 1 and SEQ ID NO:1.

The TAO1 open reading frame encodes 1001 amino acids, with a calculated molecular mass of 134 kDa. The presumed initiator codon begins at base 121 and is preceded by an in-frame stop codon at base 106. The longest 5′ UTR obtained was 600 nucleotides in length, and the longest 3′ UTR was 1200 nucleotides. None of the clones analyzed contained a poly-A track.

As is the case with most protein kinases, TAO1 can be divided into regions based on amino acid sequence comparison to other protein kinases. TAO1 is composed of an amino-terminal catalytic domain and an extensive carboxy-terminal region that has several distinguishing features, such as a possible nucleotide binding site and acidic stretch just carboxy-terminal to the catalytic domain, as well as two serine-rich regions. TAO1 does not appear to contain the leucine zipper motifs found in the MLK subfamily of kinases.

The TAO1 catalytic domain extends 263 amino acids from amino acid 25 to 288 with all 11 of the typical protein kinase subdomains conserved. There are two glutamate residues between TAO1 subdomains II and IV; the second glutamate at amino acid 76 contained in the sequence KEVK is most likely to represent subdomain III (Hanks et al., Science 241:42-52, 1988). The features of the TAO1 catalytic domain are most similar to the serine/threonine family of protein kinases; subdomain VIb with the sequence HRDIKAGN suggests that TAO1 is likely to be a serine/threonine protein kinase.

When using FASTA (GCG, Wisconsin Package) to align TAO1 with sequences from the databases, the TAO1 catalytic domain shows the highest degree of identity to a C. elegans putative serine/threonine protein kinase (accession number U32275), to which it has 63% identity and 79% similarity. That sequence appears to represent the C. elegans homolog of TAO1, and is shown as ceTAO in FIG. 2. The TAO1 catalytic domain is 39% identical to Ste20p and 40% identical to the catalytic domains of the p21-activated kinases PAK1 and PAK2. The catalytic domain of TAO1 is only 31% identical to the mixed lineage kinase MLK1, and 33% identical to dual leucine zipper-bearing kinase (DLK), also known as MLK2. Thus, TAO1 appears to be more closely related to the STE20-like kinases than to the MLK family. TAO1 is also related to germinal center kinase (GCK) and mammalian Ste20-like kinase 1 (MST1), with 42% and 45% identity respectively in the catalytic domains. The TAO1 sequence has similarity with that of the MEK kinase MEKK1. Although the overall identity between the catalytic domains of TAO1 and MEKK is only 33%, the identity of the carboxy-terminal half of their catalytic domains is higher (42%).

In the process of screening the cDNA library for clones near the 5′ end of TAO1, multiple clones representing a second closely related gene (TAO2) were identified. The TAO2 sequence is provided in SEQ ID NO:3, with the predicted amino acid sequence shown in SEQ ID NO:4 and FIG. 2. TAO2 is highly related to TAO1, and has a similar arrangement of an amino-terminal kinase domain and a long carboxy-terminus, but differs in that it contains an acidic insert of 17 glutamate residues carboxy-terminal to the catalytic domain, and lacks the putative nucleotide binding site of TAO1.

Sequences from EST databases derived from retinal mRNAs revealed the human counterpart for TAO1. The EST sequences identified are provided in SEQ ID NOs:5-16, and the alignments of these sequences with the rat TAO1 sequence are provided in FIGS. 12-20.

The Fasta program was used to compare the percent amino acid identities of several protein kinase catalytic domains, and the results are presented in Table 1, below.

TABLE 1

To assess the expression of TAO1 in transfected cells, full-length, HA-tagged TAO1 cDNA was transfected into human embryonic kidney 293 cells. A protein of approximately 140 kDa could be detected by Western blotting with an antibody directed against the HA epitope (FIG. 5A). The observed molecular mass of the protein is in good agreement with the mass predicted from the cDNA sequence.

Example 2 In vivo Expression of TAO1 and TAO2

This Example illustrates the expression of TAO1 and TAO2 in a variety of adult rat and human tissues, as determined by Northern blot analysis.

Total RNA isolated from various adult male rat tissues was selected for poly-A+ RNA with oligo(dT)cellulose (Collaborative Biomedical Products) according to the manufacturers protocols, and 5 μg of each RNA was subjected to Northern analysis. The PCR-generated 420 base fragment derived from the catalytic domain of TAO1 (described above) was labeled with [α⁻³²P]dCTP by random-priming and used to probe the Northern blot. Hybridization was at 42° C., followed by washing at 55° C. in 0.2%SSC/0.1%SDS. Integrity of the mRNA was confirmed by hybridization to an actin probe. The TAO1 probe hybridized predominately to an mRNA species of approximately 12 kb, and less strongly to another of approximately 10 kb (FIG. 3A). Of the rat tissues examined, brain clearly showed the strongest hybridization signal. On prolonged exposure, heart and lung revealed weak hybridization signals, while in skeletal muscle, liver, kidney, testis, epididymus, and spleen no signal was detected.

To assess the expression pattern of TAO2, the rat tissue Northern blot was stored until the hybridization signal for TAO1 was not seen on a two week exposure at −80° C. A fragment from the catalytic domain of TAO2 was labeled with [α⁻³²P]dCTP by random priming, and used to probe the Northern under the same hybridization and washing conditions described above for TAO1.

When the same rat tissue Northern blot was probed with a fragment of the catalytic domain of TAO2, the strongest hybridization signal was also seen in brain. The size of the transcript hybridizing to the TAO2 probe was smaller than that seen for TAO1, at 5 kb (FIG. 3B).

A probe from the non-catalytic carboxy-terminus of TAO1 (corresponding to nucleotides 1555 to 2632 of TAO1 (see FIG. 1)) was used for all additional Northern analyses because it is less likely to hybridize to TAO2 mRNA. This probe from the carboxy-terminus of TAO1 was used to assess the expression pattern in sections of human brain (Clontech). Hybridizations were performed at 68° C. in Clontech ExpressHyb buffer, and washed at 55° C. as per the manufacturer's instructions.

The strongest hybridization signals were seen in amygdala, corpus callosum, hippocampus, and substantia nigra, and each of these was stronger than that seen in whole brain (FIG. 4A). Weaker signals were seen in caudate nucleus, subthalamic nucleus and thalamus. A second human brain Northern hybridized to the same probe showed strong hybridization signals in cerebellum, putamen and occipital, frontal and temporal lobes, but much weaker signals in cerebral cortex, medulla and spinal cord (FIG. 4B).

Example 3 Kinase Activity and Substrate Specificity of TAO1

This Example illustrates the kinase activity and substrate specificity of TAO1, in in vitro and in vivo assays.

To determine whether TAO1 is active as a protein kinase, two constructs were employed. pCMV5TAO1-HA₃ and pCMV5TAO1(1-416)-HA₃ were generated by cloning the cDNAs encoding these TAO1 polypeptides into the pCMV5 mammalian expression vector. Oligonucleotide primers were used with TAO1 cDNA as template to amplify a 1247 base pair DNA product encoding amino acids 1 to 416. This fragment contains all 11 of the kinase subdomains (with the initial methionine deleted). The resulting constructs were transfected into human embryonic kidney 293 cells, and the recombinant, tagged proteins immunoprecipitated with an antibody directed against the HA epitope.

In vitro kinase assays were generally performed as follows. Kinase assays contained: 50 mM Hepes, pH 8, 10 mM MgCl₂, 1 mM DTT, 100 μM ATP, [γ-³²P]ATP (at a final concentration of 2-7 cpm/fmol), and unless otherwise noted, reactions were incubated at 30° C. for 60 minutes in a 30 μl volume. Protein kinase substrates such as myelin basic protein were added at a final concentration of 0.5 mg/ml. Reactions were halted by the addition of 10 μl 5×Laemmli buffer, followed by boiling, and 20 μl were analyzed by SDS-PAGE and autoradiography. For linked kinase assays, 50-250 ng of recombinant TAO1 protein was incubated with 50 ng of each of the bacterially expressed MEK proteins in a 30μ reaction volume for 60 minutes at 30° C., and then 5 μl of this reaction was added to a second reaction mix containing bacterially expressed (His)₆p38 or GST-SAPKβ at a final concentration of 10 μg/ml. Recombinant MEK proteins were kindly provided by Andrei Khokhlatchev and Megan Robinson, and may be prepared as described by Robinson et al., J. Biol. Chem. 271:29734-29739, 1996 and references cited therein. Within such assays, both TAO1(1-416) and full-length TAO1 were able to phosphorylate MBP in immune complex kinase reactions.

To quantitate the activity of more highly purified TAO1, TAO1(1-416), full-length TAO1 and full-length TAO1(D169A) were expressed with an amino-terminal hexa-histidine tag in Sf9 cells. TAO1(D169A) is a catalytically defective TAO1 mutant, which was created by changing aspartic acid 169 to an alanine (D169A) with PCR, and cloning the resulting construct into the pCMV5 mammalian expression vector. These constructs were prepared with either a single hemagglutinin (HA) epitope tag at the amino-terminus, a triple HA epitope tag at the carboxy-terminus, or a myc epitope tag at the amino-terminus.

The recombinant, hexa-histidine tagged TAO1, TAO1(1-416), and TAO1(D169A) were expressed in Spodoptera frugiperda (Sf9) cells. Cells were lysed by douncing in 50 mM sodium phosphate, pH 8.5, 1 mM DTT, 1 mM PMSF, and 1 mg/ml each leupeptin, pepstatin A, and aprotinin. After centrifugation at 30,000×g for 30 minutes, the supernatant was applied to a Ni²⁺-NTA agarose (Qiagen) column pre-equilibrated with the same buffer. The column was then washed with 50 column volumes of buffer, and eluted with a 20 ml gradient of 0 to 250 mM imidazole, all in the above buffer. Fractions containing recombinant TAO1 proteins were detected in fractions by Western blotting with an antibody to the MRGS(H)₆ epitope (Qiagen), and appropriate fractions were pooled and dialyzed to remove the imidazole.

(His)₆TAO1(1-416) expressed as a single 57 kDa band (FIG. 5B). Both the (His)₆TAO1 and (His)₆TAO1(D169A) recombinant proteins migrated as 140 kDa bands, although the D169A mutant appears to be more subject to degradation. (His)₆TAO1(1-416) phosphorylates MBP with a specific activity of 1 μmolmin⁻¹ mg⁻¹ in the presence of 1 mM ATP. Full-length (His)₆TAO1 exhibits MBP phosphorylating activity that is comparable to the 1-416 truncation mutant, while the activity of TAO1(D169A) is reduced to 90% of that of the wild-type protein. (His)₆TAO1(1-416) was also able to phosphorylate α-casein, histone 1, and histone 7.

To determine whether TAO1 activates one or more of the known MEKs, (His)₆TAO1(1-416) was incubated with bacterially produced MEK for one hour in the presence of Mg²⁺ and [γ⁻³²P]ATP. A portion of this reaction was then transferred to a similar reaction containing the appropriate bacterially expressed MEK substrate, (His)₆ERK2K52R for MEK1 and MEK2, (His)₆p38 for MEK3 and MEK6, and (HiS)₆P38 and GST-SAPKβ for MEK4. After a one hour incubation, the phosphoproteins were separated by SDS-PAGE. Autoradiography revealed that (His)₆TAO1(1-416) phosphorylated and activated (His)₆MEK3, and enhanced the ability of MEK3 to phosphorylate p38 by approximately 100-fold (FIG. 6).

(His)₆TAO1(1-416) activated GST-MEK4 5-fold toward (His)₆p38, and 150-fold towards GST-SAPβ (FIG. 7). The difference in fold activation seen for MEK4 towards the two substrates probably reflects the difference in basal kinase activity of MEK4 towards p38 and SAPKβ in vitro. TAO1 also increased the ability of GST-MEK6 to phosphorylate (His)₆p38, by 5-fold (FIG. 8). Recombinant GST-MEK5 was not phosphorylated by (His)₆TAO1(1-416).

Recombinant (His)₆TAO1 and (His)₆TAO1(D169A) were also examined for their ability to activate the same MEK proteins. (His)₆TAO1 showed a reduced ability to activate MEK3 as compared to that of the carboxy-terminal truncation mutant (His)₆TAO1(1-416). In multiple experiments, the full-length TAO protein displayed from 0 to 30% of the MEK3 activating ability of (His)₆TAO1(1-416), and (His)₆TAO1(D169A) was unable to activate any of the MEK proteins above basal activities.

The degree of activation of each of the MEK proteins by (His)₆TAO1(1-416) in vitro is comparable to that seen by a bacterially produced amino-terminal truncation of MEKK1 (Xu et al., Proc. Natl. Acad. Sci. USA 92:6808-6812, 1995; Robinson et al., J. Biol. Chem. 271:29734-29739, 1996). To distinguish the MEK-activating ability of TAO1 from that of MEKK, the ability of (His)₆TAO1(1-416) to activate MEK1 and MEK2 was assessed. As shown in FIG. 9, (His)₆TAO1(1-416) was completely unable to increase the activity of MEK1 or MEK2 towards the substrate (His)₆ERK2 under the same conditions that TAO1 activates MEK3, MEK4, and MEK6. Thus, while TAO1 displays MEKK-like activity in its ability to activate various MEKs, TAO1 is differentiated from MEKK by its inability to recognize MEK1 and MEK2. FIG. 9 shows the fold activation of the various MEKs by TAO1.

To assess the ability of TAO1 to activate the various MEKs in vivo, full-length HA-tagged TAO1 was co-transfected into 293 cells with myc-tagged MEK3, or myc-tagged TAO1 was co-transfected with HA-tagged MEK4 or HA-tagged MEK6. The pCMV5myc-MEK3 construct was generated by inserting the MEK3 coding sequence (provided by K. L. Guan, University of Michigan, which may be prepared as described by Robinson et al., J. Biol. Chem. 271:29734-29739, 1996) into the pCMV5Myc vector, such that the Myc epitope is at the amino-terminus of MEK3. The MEKs were then immunoprecipitated and added to immune complex kinase assays with the appropriate substrate and Mg²⁺/ATP. In multiple experiments, myc-tagged MEK3 showed a 3-fold higher activity toward p38 when immunoprecipitated from 293 cells co-expressing TAO than from cells not transfected with TAO (FIG. 10). In contrast, TAO was not able to increase the activity of immunoprecipitated HA-tagged MEK4 towards GST-SAPKβ, or that of HA-tagged MEK6 toward p38.

In transfected cells, TAO1 activates MEK3 3-fold, but neither MEK4 nor MEK6. The selectivity in transfected cells may arise from the ability of TAO1 to bind MEK3. The endogenous MEK3 from Sf9 cells copurifies with recombinant TAO1 expressed in the cells. These findings suggest that TAO1 may be an important regulator of the p38 pathway.

To determine which MEK3 residues are phosphorylated by TAO, an in vitro kinase reaction was performed with (His)₆TAO1(1-416) and (His)₆MEK3; the 57 kDa band corresponding to TAO1 and the 30 kDa band corresponding to MEK3 were excised and treated as described. Phosphoproteins were separated by SDS-PAGE, transferred to Immobilon-P membrane (Millipore) electrophoretically, and visualized by autoradiography. Bands of interest were excised and hydrolyzed in 6M HCl for 60 minutes at 110° C. The hydrolysate was dried under vacuum, and resuspended in a 2.2% formic acid, 12% acetic acid solution at an activity of 2000 cpm/μl. Then 1 μl of each sample was mixed with 1 μg each of the three phosphoamino acid standards, and spotted onto cellulose thin-layer chromatography plates. Electrophoresis was performed in 0.5% pyridine, 5% acetic acid at 1200 volts for 60 minutes. After air drying the plates, the standards were visualized with 0.25% ninhydrin in acetone. Autoradiography revealed only phosphoserine and phosphothreonine in both (His)₆TAO1(1-416) and (His)₆MEK3 (FIG. 11).

Example 4 Co-Purification of MEK3 and TAO1

This Example shows that TAO1 and MEK3 co-purify.

Although the ability of (His)₆TAO1 to activate MEK3 was always reduced in comparison with that of (His)₆TAO1(1-416), several assays showed that the ability of (His)₆TAO1 to lead to an increase in the phosphorylation of p38 in the linked kinase assays was partly independent of the addition of MEK. (His)₆TAO1(1-416) does not phosphorylate p38. Therefore, Western analyses were performed to determine if one or more MEKs might be present in the TAO1 preparations purified from Sf9 cells.

(His)₆TAO1, (His)₆TAO1 (1-416), and (His)₆TAO1(D169A) were subjected to Western analysis with antisera specific to MEK3, MEK4, and MEK6. Four different polyclonal antisera were raised to these three TAO1 peptides in rabbits. The peptide TKDAVRELDNLQYRKMKKLL (SEQ ID NO:23) corresponding to the amino acids 296 to 315 yielded antisera P820. The peptide KKELNSFLESQKREYKLRK (SEQ ID NO:24) of amino acids 545 to 563 yielded the antiserum R562. Finally, the peptide RELRELEQRVSLRRALLEQK (SEQ ID NO:25) of amino acids 829 to 848 resulted in the antisera R564 and R565. These peptides were conjugated to Limulus hemocyanin (Boulton and Cobb, Cell. Regul. 2:357-371, 1991) and dialyzed into phosphate-buffered saline. A total of five boosts were performed, after which the rabbits were exsanguinated and the serum collected. The antisera were screened for reactivity by Western blotting of recombinant TAO1 expressed in Sf9 cells. Five antisera were found to consistently recognize the recombinant TAO1 protein in Western blots. Free peptide was able to block the specific recognition of TAO1 protein by the antisera. None of the five antisera detected the presence of TAO1 in lysates of 293, NIH3T3, NG-108, or COS cells.

For immunoblot analysis, either 50 ng of recombinant TAO1 protein or 100 μg of cell lysate was subjected to SDS-PAGE, then transferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat powdered milk in TBST (20 mM Tris, pH 8, 500 mM NaCl, 0.05% Tween 20) for one hour, then incubated with the polyclonal antisera at 1:500 dilution in TBST plus 0.25% milk for one hour. After three washes with TBST, the membranes were incubated with a 1:2500 dilution of horseradish peroxidase-conjugated goat-anti-rabbit IgG in TBST plus 0.25% milk for one hour. Membranes were washed again in TBST then visualized with the ECL system (Amersham).

MEK3 was clearly seen in the (His)₆TAO1 preparation, and to a lesser extent in the (His)₆TAO1(D169A) preparation (FIG. 11). MEK4 was detected in the Sf9 cell lysates, but not in the TAO1 preparations, while MEK6 was detected in neither.

From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for the purpose of illustration, various modifications may be made without deviating from the spirit and scope of the invention.

26 3312 base pairs nucleic acid single linear CDS 121..3123 1 TCTGCAGTAT GGTAGATTAT TATTTATGCA TTTATGCCAG TGTGGCTTCA TTCATACAGA 60 TGAACCAAGC TTTGGGATAG CAGTATAAAA TTAGAATCAG ACAGCTGACT GCTCAGCAGG 120 ATG CCA TCA ACT AAC AGA GCA GGC AGT CTA AAG GAC CCT GAA ATC GCA 168 Met Pro Ser Thr Asn Arg Ala Gly Ser Leu Lys Asp Pro Glu Ile Ala 1 5 10 15 GAG CTC TTC TTC AAA GAA GAT CCG GAA AAA CTC TTC ACA GAT CTC AGA 216 Glu Leu Phe Phe Lys Glu Asp Pro Glu Lys Leu Phe Thr Asp Leu Arg 20 25 30 GAA ATC GGC CAT GGG AGC TTT GGA GCA GTT TAT TTT GCA CGA GAT GTG 264 Glu Ile Gly His Gly Ser Phe Gly Ala Val Tyr Phe Ala Arg Asp Val 35 40 45 CGT ACT AAT GAA GTG GTG GCC ATC AAG AAA ATG TCT TAT AGT GGA AAG 312 Arg Thr Asn Glu Val Val Ala Ile Lys Lys Met Ser Tyr Ser Gly Lys 50 55 60 CAG TCT ACT GAG AAA TGG CAG GAT ATT ATT AAG GAA GTC AAG TTT CTA 360 Gln Ser Thr Glu Lys Trp Gln Asp Ile Ile Lys Glu Val Lys Phe Leu 65 70 75 80 CAA AGA ATA AAA CAT CCC AAC AGT ATA GAA TAC AAA GGC TGC TAT TTA 408 Gln Arg Ile Lys His Pro Asn Ser Ile Glu Tyr Lys Gly Cys Tyr Leu 85 90 95 CGT GAA CAC ACA GCA TGG CTT GTA ATG GAA TAT TGT TTA GGA TCT GCT 456 Arg Glu His Thr Ala Trp Leu Val Met Glu Tyr Cys Leu Gly Ser Ala 100 105 110 TCG GAT TTA CTA GAA GTT CAT AAA AAG CCA TTA CAA GAA GTG GAA ATA 504 Ser Asp Leu Leu Glu Val His Lys Lys Pro Leu Gln Glu Val Glu Ile 115 120 125 GCA GCA ATT ACA CAT GGT GCT CTC CAG GGA TTA GCT TAT TTA CAT TCT 552 Ala Ala Ile Thr His Gly Ala Leu Gln Gly Leu Ala Tyr Leu His Ser 130 135 140 CAT ACC ATG ATC CAT AGA GAT ATC AAA GCA GGA AAT ATC CTT CTG ACA 600 His Thr Met Ile His Arg Asp Ile Lys Ala Gly Asn Ile Leu Leu Thr 145 150 155 160 GAA CCA GGC CAA GTG AAA CTT GCT GAC TTT GGA TCT GCT TCC ATG GCC 648 Glu Pro Gly Gln Val Lys Leu Ala Asp Phe Gly Ser Ala Ser Met Ala 165 170 175 TCC CCT GCC AAT TCT TTT GTG GGA ACA CCA TAT TGG ATG GCC CCA GAA 696 Ser Pro Ala Asn Ser Phe Val Gly Thr Pro Tyr Trp Met Ala Pro Glu 180 185 190 GTA ATT TTA GCC ATG GAT GAA GGA CAA TAT GAT GGC AAA GTT GAT GTA 744 Val Ile Leu Ala Met Asp Glu Gly Gln Tyr Asp Gly Lys Val Asp Val 195 200 205 TGG TCT CTT GGA ATA ACA TGT ATT GAA TTA GCC GAG AGG AAG CCT CCT 792 Trp Ser Leu Gly Ile Thr Cys Ile Glu Leu Ala Glu Arg Lys Pro Pro 210 215 220 TTA TTT AAT ATG AAT GCA ATG AGT GCC TTA TAT CAC ATA GCC CAA AAT 840 Leu Phe Asn Met Asn Ala Met Ser Ala Leu Tyr His Ile Ala Gln Asn 225 230 235 240 GAA TCC CCT ACA CTA CAG TCT AAT GAA TGG TCT GAT TAT TTT CGA AAC 888 Glu Ser Pro Thr Leu Gln Ser Asn Glu Trp Ser Asp Tyr Phe Arg Asn 245 250 255 TTT GTA GAT TCT TGC CTC CAG AAA ATC CCT CAA GAT CGC CCT ACA TCA 936 Phe Val Asp Ser Cys Leu Gln Lys Ile Pro Gln Asp Arg Pro Thr Ser 260 265 270 GAG GAA CTT TTA AAG CAC ATG TTT GTT CTT CGA GAG CGC CCT GAA ACA 984 Glu Glu Leu Leu Lys His Met Phe Val Leu Arg Glu Arg Pro Glu Thr 275 280 285 GTG TTA ATA GAT CTT ATT CAA AGG ACA AAG GAT GCA GTA AGA GAG CTG 1032 Val Leu Ile Asp Leu Ile Gln Arg Thr Lys Asp Ala Val Arg Glu Leu 290 295 300 GAC AAT CTA CAA TAT CGA AAG ATG AAG AAA CTC CTT TTC CAG GAG GCA 1080 Asp Asn Leu Gln Tyr Arg Lys Met Lys Lys Leu Leu Phe Gln Glu Ala 305 310 315 320 CAT AAT GGA CCA GCA GTA GAA GCA CAG GAA GAA GAG GAG GAG CAA GAT 1128 His Asn Gly Pro Ala Val Glu Ala Gln Glu Glu Glu Glu Glu Gln Asp 325 330 335 CAT GGT GGT GGC CGG ACA GGA ACA GTA AAT AGT GTT GGA AGC AAT CAG 1176 His Gly Gly Gly Arg Thr Gly Thr Val Asn Ser Val Gly Ser Asn Gln 340 345 350 TCT ATC CCC AGT ATG TCT ATC AGT GCC AGT AGC CAA AGC AGC AGT GTT 1224 Ser Ile Pro Ser Met Ser Ile Ser Ala Ser Ser Gln Ser Ser Ser Val 355 360 365 AAT AGT CTT CCA GAT GCA TCG GAT GAC AAG AGT GAG CTA GAC ATG ATG 1272 Asn Ser Leu Pro Asp Ala Ser Asp Asp Lys Ser Glu Leu Asp Met Met 370 375 380 GAG GGA GAC CAT ACA GTG ATG TCT AAC AGT TCT GTC ATC CAC TTA AAA 1320 Glu Gly Asp His Thr Val Met Ser Asn Ser Ser Val Ile His Leu Lys 385 390 395 400 CCT GAG GAG GAA AAT TAC CAA GAA GAA GGA GAT CCT AGA ACA AGA GCA 1368 Pro Glu Glu Glu Asn Tyr Gln Glu Glu Gly Asp Pro Arg Thr Arg Ala 405 410 415 TCA GCT CCA CAG TCT CCA CCT CAA GTG TCT CGT CAC AAA TCA CAT TAT 1416 Ser Ala Pro Gln Ser Pro Pro Gln Val Ser Arg His Lys Ser His Tyr 420 425 430 CGT AAT AGA GAA CAC TTT GCA ACT ATA CGA ACA GCA TCA CTG GTT ACA 1464 Arg Asn Arg Glu His Phe Ala Thr Ile Arg Thr Ala Ser Leu Val Thr 435 440 445 AGA CAG ATG CAA GAA CAT GAG CAG GAC TCT GAA CTT AGA GAA CAG ATG 1512 Arg Gln Met Gln Glu His Glu Gln Asp Ser Glu Leu Arg Glu Gln Met 450 455 460 TCT GGT TAT AAG CGG ATG AGG CGA CAG CAT CAG AAG CAG CTG ATG ACT 1560 Ser Gly Tyr Lys Arg Met Arg Arg Gln His Gln Lys Gln Leu Met Thr 465 470 475 480 CTG GAA AAT AAA CTG AAG GCA GAA ATG GAC GAA CAT CGG CTC AGA TTA 1608 Leu Glu Asn Lys Leu Lys Ala Glu Met Asp Glu His Arg Leu Arg Leu 485 490 495 GAC AAA GAT CTT GAA ACT CAG CGC AAC AAT TTC GCT GCA GAA ATG GAG 1656 Asp Lys Asp Leu Glu Thr Gln Arg Asn Asn Phe Ala Ala Glu Met Glu 500 505 510 AAA CTT ATT AAG AAA CAC CAA GCT TCT ATG GAA AAA GAG GCT AAA GTG 1704 Lys Leu Ile Lys Lys His Gln Ala Ser Met Glu Lys Glu Ala Lys Val 515 520 525 ATG GCC AAC GAG GAG AAA AAA TTC CAA CAA CAC ATT CAG GCT CAA CAG 1752 Met Ala Asn Glu Glu Lys Lys Phe Gln Gln His Ile Gln Ala Gln Gln 530 535 540 AAG AAA GAA CTG AAT AGC TTT TTG GAG TCT CAA AAA AGA GAA TAT AAA 1800 Lys Lys Glu Leu Asn Ser Phe Leu Glu Ser Gln Lys Arg Glu Tyr Lys 545 550 555 560 CTT CGA AAA GAG CAG CTT AAG GAG GAG CTG AAT GAA AAC CAG AGC ACA 1848 Leu Arg Lys Glu Gln Leu Lys Glu Glu Leu Asn Glu Asn Gln Ser Thr 565 570 575 CCT AAA AAA GAA AAG CAG GAA TGG CTT TCA AAG CAG AAG GAG AAT ATT 1896 Pro Lys Lys Glu Lys Gln Glu Trp Leu Ser Lys Gln Lys Glu Asn Ile 580 585 590 CAA CAT TTT CAG GCA GAA GAA GAA GCT AAT CTT CTT CGA CGT CAA AGG 1944 Gln His Phe Gln Ala Glu Glu Glu Ala Asn Leu Leu Arg Arg Gln Arg 595 600 605 CAG TAT CTA GAG CTA GAA TGT CGT CGC TTC AAA AGA AGA ATG TTA CTT 1992 Gln Tyr Leu Glu Leu Glu Cys Arg Arg Phe Lys Arg Arg Met Leu Leu 610 615 620 GGT CGG CAT AAC TTG GAA CAG GAC CTT GTC AGG GAG GAG TTA AAC AAA 2040 Gly Arg His Asn Leu Glu Gln Asp Leu Val Arg Glu Glu Leu Asn Lys 625 630 635 640 AGG CAG ACT CAG AAG GAC TTA GAA CAT GCA ATG TTA CTG CGA CAG CAT 2088 Arg Gln Thr Gln Lys Asp Leu Glu His Ala Met Leu Leu Arg Gln His 645 650 655 GAA TCC ATG CAA GAA CTG GAG TTT CGC CAC CTC AAC ACT ATT CAG AAG 2136 Glu Ser Met Gln Glu Leu Glu Phe Arg His Leu Asn Thr Ile Gln Lys 660 665 670 ATG CGC TGT GAG TTG ATC AGA CTG CAA CAT CAA ACT GAG CTT ACT AAC 2184 Met Arg Cys Glu Leu Ile Arg Leu Gln His Gln Thr Glu Leu Thr Asn 675 680 685 CAG CTG GAA TAC AAT AAG AGA AGG GAA CGG GAA CTA AGA CGG AAA CAT 2232 Gln Leu Glu Tyr Asn Lys Arg Arg Glu Arg Glu Leu Arg Arg Lys His 690 695 700 GTC ATG GAA GTT CGA CAG CAG CCT AAG AGT TTG AAG TCT AAA GAA CTC 2280 Val Met Glu Val Arg Gln Gln Pro Lys Ser Leu Lys Ser Lys Glu Leu 705 710 715 720 CAA ATA AAA AAG CAG TTT CAG GAT ACC TGC AAA ATT CAA ACC AGA CAG 2328 Gln Ile Lys Lys Gln Phe Gln Asp Thr Cys Lys Ile Gln Thr Arg Gln 725 730 735 TAC AAA GCA TTA AGG AAT CAC CTA CTG GAG ACT ACA CCA AAG AGT GAG 2376 Tyr Lys Ala Leu Arg Asn His Leu Leu Glu Thr Thr Pro Lys Ser Glu 740 745 750 CAC AAA GCT GTT CTG AAA AGA CTC AAG GAG GAA CAG ACT CGG AAG TTA 2424 His Lys Ala Val Leu Lys Arg Leu Lys Glu Glu Gln Thr Arg Lys Leu 755 760 765 GCC ATC TTG GCT GAG CAG TAT GAT CAT AGC ATT AAT GAA ATG CTC TCC 2472 Ala Ile Leu Ala Glu Gln Tyr Asp His Ser Ile Asn Glu Met Leu Ser 770 775 780 ACA CAA GCT CTG CGT TTG GAT GAA GCA CAG GAA GCA GAA TGC CAG GTT 2520 Thr Gln Ala Leu Arg Leu Asp Glu Ala Gln Glu Ala Glu Cys Gln Val 785 790 795 800 TTG AAG ATG CAG CTA CAG CAG GAA CTG GAG CTG TTG AAT GCA TAT CAG 2568 Leu Lys Met Gln Leu Gln Gln Glu Leu Glu Leu Leu Asn Ala Tyr Gln 805 810 815 AGC AAA ATC AAG ATG CAG GCT GAG GCC CAA CAT GAT CGA GAG CTT CGA 2616 Ser Lys Ile Lys Met Gln Ala Glu Ala Gln His Asp Arg Glu Leu Arg 820 825 830 GAG CTG GAA CAA AGG GTC TCC CTT CGG AGA GCA CTC TTA GAA CAG AAG 2664 Glu Leu Glu Gln Arg Val Ser Leu Arg Arg Ala Leu Leu Glu Gln Lys 835 840 845 ATT GAA GAA GAG ATG TTG GCT TTG CAG AAT GAA CGC ACA GAA CGA ATA 2712 Ile Glu Glu Glu Met Leu Ala Leu Gln Asn Glu Arg Thr Glu Arg Ile 850 855 860 CGT AGC CTG CTC GAG CGC CAG GCC AGA GAA ATT GAA GCT TTT GAC TCT 2760 Arg Ser Leu Leu Glu Arg Gln Ala Arg Glu Ile Glu Ala Phe Asp Ser 865 870 875 880 GAA AGC ATG AGA TTA GGT TTT AGT AAC ATG GTC CTT TCT AAT CTC TCC 2808 Glu Ser Met Arg Leu Gly Phe Ser Asn Met Val Leu Ser Asn Leu Ser 885 890 895 CCT GAG GCA TTC AGC CAC AGC TAC CCA GGA GCT TCT AGC TGG TCT CAC 2856 Pro Glu Ala Phe Ser His Ser Tyr Pro Gly Ala Ser Ser Trp Ser His 900 905 910 AAT CCT ACT GGG GGT TCA GGA CCT CAC TGG GGT CAT CCC ATG GGT GGC 2904 Asn Pro Thr Gly Gly Ser Gly Pro His Trp Gly His Pro Met Gly Gly 915 920 925 ACA CCA CAA GCT TGG GGT CAT CCG ATG CAA GGC GGA CCC CAA CCA TGG 2952 Thr Pro Gln Ala Trp Gly His Pro Met Gln Gly Gly Pro Gln Pro Trp 930 935 940 GGT CAC CCC TCA GGG CCA ATG CAA GGG GTA CCT CGA GGT AGC AGT ATA 3000 Gly His Pro Ser Gly Pro Met Gln Gly Val Pro Arg Gly Ser Ser Ile 945 950 955 960 GGA GTC CGC AAT AGC CCC CAG GCT CTG AGG CGG ACA GCT TCT GGG GGA 3048 Gly Val Arg Asn Ser Pro Gln Ala Leu Arg Arg Thr Ala Ser Gly Gly 965 970 975 CGG ACG GAA CAG GGC ATG AGC AGA AGC ACG AGT GTC ACT TCA CAA ATA 3096 Arg Thr Glu Gln Gly Met Ser Arg Ser Thr Ser Val Thr Ser Gln Ile 980 985 990 TCC AAT GGG TCA CAC ATG TCT TAC ACA TAATAATTGA AAGTGGCAAT 3143 Ser Asn Gly Ser His Met Ser Tyr Thr 995 1000 TCCGCTGGAG CTGTCTGCCA AAAGAAACTG CCTACAGACA TCAGCACAGC AGCCTCCTCA 3203 CTTGGGTACT ACCGGGTGGA AGCTGTGCAT ATGGTATATT TTATTCGTCT TTGTAAAGCG 3263 TTATGTTTTG TGTTTACTAA TTGGGATGTC ATAGTATTTG GCTGCCGGG 3312 1001 amino acids amino acid linear protein 2 Met Pro Ser Thr Asn Arg Ala Gly Ser Leu Lys Asp Pro Glu Ile Ala 1 5 10 15 Glu Leu Phe Phe Lys Glu Asp Pro Glu Lys Leu Phe Thr Asp Leu Arg 20 25 30 Glu Ile Gly His Gly Ser Phe Gly Ala Val Tyr Phe Ala Arg Asp Val 35 40 45 Arg Thr Asn Glu Val Val Ala Ile Lys Lys Met Ser Tyr Ser Gly Lys 50 55 60 Gln Ser Thr Glu Lys Trp Gln Asp Ile Ile Lys Glu Val Lys Phe Leu 65 70 75 80 Gln Arg Ile Lys His Pro Asn Ser Ile Glu Tyr Lys Gly Cys Tyr Leu 85 90 95 Arg Glu His Thr Ala Trp Leu Val Met Glu Tyr Cys Leu Gly Ser Ala 100 105 110 Ser Asp Leu Leu Glu Val His Lys Lys Pro Leu Gln Glu Val Glu Ile 115 120 125 Ala Ala Ile Thr His Gly Ala Leu Gln Gly Leu Ala Tyr Leu His Ser 130 135 140 His Thr Met Ile His Arg Asp Ile Lys Ala Gly Asn Ile Leu Leu Thr 145 150 155 160 Glu Pro Gly Gln Val Lys Leu Ala Asp Phe Gly Ser Ala Ser Met Ala 165 170 175 Ser Pro Ala Asn Ser Phe Val Gly Thr Pro Tyr Trp Met Ala Pro Glu 180 185 190 Val Ile Leu Ala Met Asp Glu Gly Gln Tyr Asp Gly Lys Val Asp Val 195 200 205 Trp Ser Leu Gly Ile Thr Cys Ile Glu Leu Ala Glu Arg Lys Pro Pro 210 215 220 Leu Phe Asn Met Asn Ala Met Ser Ala Leu Tyr His Ile Ala Gln Asn 225 230 235 240 Glu Ser Pro Thr Leu Gln Ser Asn Glu Trp Ser Asp Tyr Phe Arg Asn 245 250 255 Phe Val Asp Ser Cys Leu Gln Lys Ile Pro Gln Asp Arg Pro Thr Ser 260 265 270 Glu Glu Leu Leu Lys His Met Phe Val Leu Arg Glu Arg Pro Glu Thr 275 280 285 Val Leu Ile Asp Leu Ile Gln Arg Thr Lys Asp Ala Val Arg Glu Leu 290 295 300 Asp Asn Leu Gln Tyr Arg Lys Met Lys Lys Leu Leu Phe Gln Glu Ala 305 310 315 320 His Asn Gly Pro Ala Val Glu Ala Gln Glu Glu Glu Glu Glu Gln Asp 325 330 335 His Gly Gly Gly Arg Thr Gly Thr Val Asn Ser Val Gly Ser Asn Gln 340 345 350 Ser Ile Pro Ser Met Ser Ile Ser Ala Ser Ser Gln Ser Ser Ser Val 355 360 365 Asn Ser Leu Pro Asp Ala Ser Asp Asp Lys Ser Glu Leu Asp Met Met 370 375 380 Glu Gly Asp His Thr Val Met Ser Asn Ser Ser Val Ile His Leu Lys 385 390 395 400 Pro Glu Glu Glu Asn Tyr Gln Glu Glu Gly Asp Pro Arg Thr Arg Ala 405 410 415 Ser Ala Pro Gln Ser Pro Pro Gln Val Ser Arg His Lys Ser His Tyr 420 425 430 Arg Asn Arg Glu His Phe Ala Thr Ile Arg Thr Ala Ser Leu Val Thr 435 440 445 Arg Gln Met Gln Glu His Glu Gln Asp Ser Glu Leu Arg Glu Gln Met 450 455 460 Ser Gly Tyr Lys Arg Met Arg Arg Gln His Gln Lys Gln Leu Met Thr 465 470 475 480 Leu Glu Asn Lys Leu Lys Ala Glu Met Asp Glu His Arg Leu Arg Leu 485 490 495 Asp Lys Asp Leu Glu Thr Gln Arg Asn Asn Phe Ala Ala Glu Met Glu 500 505 510 Lys Leu Ile Lys Lys His Gln Ala Ser Met Glu Lys Glu Ala Lys Val 515 520 525 Met Ala Asn Glu Glu Lys Lys Phe Gln Gln His Ile Gln Ala Gln Gln 530 535 540 Lys Lys Glu Leu Asn Ser Phe Leu Glu Ser Gln Lys Arg Glu Tyr Lys 545 550 555 560 Leu Arg Lys Glu Gln Leu Lys Glu Glu Leu Asn Glu Asn Gln Ser Thr 565 570 575 Pro Lys Lys Glu Lys Gln Glu Trp Leu Ser Lys Gln Lys Glu Asn Ile 580 585 590 Gln His Phe Gln Ala Glu Glu Glu Ala Asn Leu Leu Arg Arg Gln Arg 595 600 605 Gln Tyr Leu Glu Leu Glu Cys Arg Arg Phe Lys Arg Arg Met Leu Leu 610 615 620 Gly Arg His Asn Leu Glu Gln Asp Leu Val Arg Glu Glu Leu Asn Lys 625 630 635 640 Arg Gln Thr Gln Lys Asp Leu Glu His Ala Met Leu Leu Arg Gln His 645 650 655 Glu Ser Met Gln Glu Leu Glu Phe Arg His Leu Asn Thr Ile Gln Lys 660 665 670 Met Arg Cys Glu Leu Ile Arg Leu Gln His Gln Thr Glu Leu Thr Asn 675 680 685 Gln Leu Glu Tyr Asn Lys Arg Arg Glu Arg Glu Leu Arg Arg Lys His 690 695 700 Val Met Glu Val Arg Gln Gln Pro Lys Ser Leu Lys Ser Lys Glu Leu 705 710 715 720 Gln Ile Lys Lys Gln Phe Gln Asp Thr Cys Lys Ile Gln Thr Arg Gln 725 730 735 Tyr Lys Ala Leu Arg Asn His Leu Leu Glu Thr Thr Pro Lys Ser Glu 740 745 750 His Lys Ala Val Leu Lys Arg Leu Lys Glu Glu Gln Thr Arg Lys Leu 755 760 765 Ala Ile Leu Ala Glu Gln Tyr Asp His Ser Ile Asn Glu Met Leu Ser 770 775 780 Thr Gln Ala Leu Arg Leu Asp Glu Ala Gln Glu Ala Glu Cys Gln Val 785 790 795 800 Leu Lys Met Gln Leu Gln Gln Glu Leu Glu Leu Leu Asn Ala Tyr Gln 805 810 815 Ser Lys Ile Lys Met Gln Ala Glu Ala Gln His Asp Arg Glu Leu Arg 820 825 830 Glu Leu Glu Gln Arg Val Ser Leu Arg Arg Ala Leu Leu Glu Gln Lys 835 840 845 Ile Glu Glu Glu Met Leu Ala Leu Gln Asn Glu Arg Thr Glu Arg Ile 850 855 860 Arg Ser Leu Leu Glu Arg Gln Ala Arg Glu Ile Glu Ala Phe Asp Ser 865 870 875 880 Glu Ser Met Arg Leu Gly Phe Ser Asn Met Val Leu Ser Asn Leu Ser 885 890 895 Pro Glu Ala Phe Ser His Ser Tyr Pro Gly Ala Ser Ser Trp Ser His 900 905 910 Asn Pro Thr Gly Gly Ser Gly Pro His Trp Gly His Pro Met Gly Gly 915 920 925 Thr Pro Gln Ala Trp Gly His Pro Met Gln Gly Gly Pro Gln Pro Trp 930 935 940 Gly His Pro Ser Gly Pro Met Gln Gly Val Pro Arg Gly Ser Ser Ile 945 950 955 960 Gly Val Arg Asn Ser Pro Gln Ala Leu Arg Arg Thr Ala Ser Gly Gly 965 970 975 Arg Thr Glu Gln Gly Met Ser Arg Ser Thr Ser Val Thr Ser Gln Ile 980 985 990 Ser Asn Gly Ser His Met Ser Tyr Thr 995 1000 4296 base pairs nucleic acid single linear CDS 193..3171 3 AGGGGAGGCT TCCCGGGCCC GCCCCTCAGG AAGGGCGAAA GCTGAGGAAG AGGTGGCGAG 60 GGGGAAGGTC TCCTTGCCCC TCTCCCCGCT TGTCAGAGCA ACTGGAGTAC CCCAGGCGGA 120 AGCGGAGGCG CTGGGGCACC ATAGTGACCC CTACCAGGCA AGATCCCAAT TTCAGGGCCC 180 CCAGGGGCCA TC ATG CCA GCT GGG GGC CGG GCC GGG AGC CTG AAG GAC 228 Met Pro Ala Gly Gly Arg Ala Gly Ser Leu Lys Asp 1 5 10 CCT GAT GTA GCT GAG CTC TTC TTC AAA GAT GAC CCT GAG AAG CTT TTC 276 Pro Asp Val Ala Glu Leu Phe Phe Lys Asp Asp Pro Glu Lys Leu Phe 15 20 25 TCT GAC CTC CGG GAA ATT GGC CAT GGC AGT TTT GGA GCT GTG TAC TTT 324 Ser Asp Leu Arg Glu Ile Gly His Gly Ser Phe Gly Ala Val Tyr Phe 30 35 40 GCC CGG GAT GTC CGG AAC AGT GAG GTG GTG GCC ATC AAG AAG ATG TCC 372 Ala Arg Asp Val Arg Asn Ser Glu Val Val Ala Ile Lys Lys Met Ser 45 50 55 60 TAT AGT GGG AAG CAA TCA AAT GAG AAA TGG CAG GAT ATC ATC AAG GAG 420 Tyr Ser Gly Lys Gln Ser Asn Glu Lys Trp Gln Asp Ile Ile Lys Glu 65 70 75 GTG CGG TTC TTA CAG AAG CTA CGG CAT CCT AAT ACC ATT CAG TAC CGG 468 Val Arg Phe Leu Gln Lys Leu Arg His Pro Asn Thr Ile Gln Tyr Arg 80 85 90 GGC TGT TAC CTG AGG GAG CAC ACA GCT TGG CTG GTG ATG GAG TAT TGC 516 Gly Cys Tyr Leu Arg Glu His Thr Ala Trp Leu Val Met Glu Tyr Cys 95 100 105 CTG GGT TCA GCT TCT GAT CTT CTC GAA GTG CAC AAG AAG CCG CTG CAG 564 Leu Gly Ser Ala Ser Asp Leu Leu Glu Val His Lys Lys Pro Leu Gln 110 115 120 GAG GTA GAG ATT GCA GCT GTG ACC CAT GGT GCG CTT CAG GGC CTG GCC 612 Glu Val Glu Ile Ala Ala Val Thr His Gly Ala Leu Gln Gly Leu Ala 125 130 135 140 TAT CTA CAT TCA CAC AAC ATG ATC CAT AGA GAT GTG AAG GCT GGG AAC 660 Tyr Leu His Ser His Asn Met Ile His Arg Asp Val Lys Ala Gly Asn 145 150 155 ATC TTG CTG TCA GAA CCA GGC TTG GTG AAA CTG GGG GAC TTT GGC TCC 708 Ile Leu Leu Ser Glu Pro Gly Leu Val Lys Leu Gly Asp Phe Gly Ser 160 165 170 GCA TCC ATC ATG GCA CCT GCC AAC TCA TTT GTG GGC ACT CCA TAC TGG 756 Ala Ser Ile Met Ala Pro Ala Asn Ser Phe Val Gly Thr Pro Tyr Trp 175 180 185 ATG GCT CCA GAG GTG ATC CTA GCC ATG GAT GAG GGA CAA TAT GAT GGC 804 Met Ala Pro Glu Val Ile Leu Ala Met Asp Glu Gly Gln Tyr Asp Gly 190 195 200 AAA GTG GAT GTC TGG TCC TTG GGG ATA ACC TGT ATT GAG CTA GCG GAG 852 Lys Val Asp Val Trp Ser Leu Gly Ile Thr Cys Ile Glu Leu Ala Glu 205 210 215 220 CGG AAG CCA CCA CTG TTT AAC ATG AAT GCA ATG AGT GCC TTA TAC CAC 900 Arg Lys Pro Pro Leu Phe Asn Met Asn Ala Met Ser Ala Leu Tyr His 225 230 235 ATT GCA CAG AAT GAA TCC CCT GCT CTC CAG TCA GGA CAC TGG TCT GAG 948 Ile Ala Gln Asn Glu Ser Pro Ala Leu Gln Ser Gly His Trp Ser Glu 240 245 250 TAC TTC CGG AAT TTT GTT GAC TCC TGT CTT CAG AAA ATC CCT CAA GAC 996 Tyr Phe Arg Asn Phe Val Asp Ser Cys Leu Gln Lys Ile Pro Gln Asp 255 260 265 AGA CCA ACC TCA GAG GTT CTT TTG AAG CAC CGC TTT GTG CTC CGG GAG 1044 Arg Pro Thr Ser Glu Val Leu Leu Lys His Arg Phe Val Leu Arg Glu 270 275 280 CGG CCA CCC ACA GTC ATC ATG GAC CTA ATT CAG AGG ACC AAG GAT GCT 1092 Arg Pro Pro Thr Val Ile Met Asp Leu Ile Gln Arg Thr Lys Asp Ala 285 290 295 300 GTA CGG GAA CTA GAT AAC CTG CAG TAC CGA AAG ATG AAG AAG ATA CTA 1140 Val Arg Glu Leu Asp Asn Leu Gln Tyr Arg Lys Met Lys Lys Ile Leu 305 310 315 TTC CAA GAG GCA CCC AAT GGC CCT GGT GCT GAG GCC CCA GAG GAA GAG 1188 Phe Gln Glu Ala Pro Asn Gly Pro Gly Ala Glu Ala Pro Glu Glu Glu 320 325 330 GAG GAA GCA GAA CCT TAC ATG CAC CGA GCA GGG ACA CTG ACC AGT CTA 1236 Glu Glu Ala Glu Pro Tyr Met His Arg Ala Gly Thr Leu Thr Ser Leu 335 340 345 GAG AGT AGC CAT TCA GTG CCC AGC ATG TCC ATC AGC GCC TCC AGC CAA 1284 Glu Ser Ser His Ser Val Pro Ser Met Ser Ile Ser Ala Ser Ser Gln 350 355 360 AGC AGC TCA GTC AAC AGC CTA GCA GAT GCC TCA GAT AAT GAA GAA GAG 1332 Ser Ser Ser Val Asn Ser Leu Ala Asp Ala Ser Asp Asn Glu Glu Glu 365 370 375 380 GAG GAG GAG GAA GAG GAA GAA GAA GAG GAG GAG GAA GAA GAA GGC CCT 1380 Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Gly Pro 385 390 395 GAA TCC CGA GAG ATG GCC ATG ATG CAG GAG GGG GAG CAT ACA GTC ACT 1428 Glu Ser Arg Glu Met Ala Met Met Gln Glu Gly Glu His Thr Val Thr 400 405 410 TCC CAC AGC TCC ATC ATC CAC CGG CTG CCG GGC TCA GAC AAC CTA TAT 1476 Ser His Ser Ser Ile Ile His Arg Leu Pro Gly Ser Asp Asn Leu Tyr 415 420 425 GAT GAT CCC TAC CAG CCA GAG ATG ACC CCA GGT CCA CTC CAA CCA CCT 1524 Asp Asp Pro Tyr Gln Pro Glu Met Thr Pro Gly Pro Leu Gln Pro Pro 430 435 440 GCA GCC CCT CCC ACC TCC ACC TCC TCC TCT TCT GCT CGC CGC AGA GCT 1572 Ala Ala Pro Pro Thr Ser Thr Ser Ser Ser Ser Ala Arg Arg Arg Ala 445 450 455 460 TAT TGC CGC AAC CGA GAC CAC TTT GCC ACC ATC CGT ACT GCC TCC CTG 1620 Tyr Cys Arg Asn Arg Asp His Phe Ala Thr Ile Arg Thr Ala Ser Leu 465 470 475 GTC AGC CGT CAG ATC CAG GAG CAT GAG CAG GAC TCG GCC CTG CGG GAG 1668 Val Ser Arg Gln Ile Gln Glu His Glu Gln Asp Ser Ala Leu Arg Glu 480 485 490 CAA CTA AGT GGC TAC AAG CGG ATG CGG CGT CAG CAC CAG AAG CAA CTG 1716 Gln Leu Ser Gly Tyr Lys Arg Met Arg Arg Gln His Gln Lys Gln Leu 495 500 505 CTG GCC CTG GAG TCC CGT CTG AGG GGT GAA CGT GAG GAG CAC AGT GGG 1764 Leu Ala Leu Glu Ser Arg Leu Arg Gly Glu Arg Glu Glu His Ser Gly 510 515 520 CGG TTG CAG CGT GAA CTC GAG GCA CAG CGG GCT GGC TTT GGG ACT GAG 1812 Arg Leu Gln Arg Glu Leu Glu Ala Gln Arg Ala Gly Phe Gly Thr Glu 525 530 535 540 GCT GAG AAG CTG GCC CGG AGG CAC CAG GCC ATT GGT GAG AAG GAA GCA 1860 Ala Glu Lys Leu Ala Arg Arg His Gln Ala Ile Gly Glu Lys Glu Ala 545 550 555 CGA GCT GCT CAG GCT GAG GAG CGG AAG TTC CAG CAG CAC ATC TTG GGG 1908 Arg Ala Ala Gln Ala Glu Glu Arg Lys Phe Gln Gln His Ile Leu Gly 560 565 570 CAG CAG AAG AAG GAA CTG GCT GCC CTG CTG GAG GCA CAG AAG CGA ACC 1956 Gln Gln Lys Lys Glu Leu Ala Ala Leu Leu Glu Ala Gln Lys Arg Thr 575 580 585 TAT AAG CTT CGG AAG GAG CAG TTG AAA GAG GAG CTC CAG GAG AAC CCT 2004 Tyr Lys Leu Arg Lys Glu Gln Leu Lys Glu Glu Leu Gln Glu Asn Pro 590 595 600 AGC ACA CCC AAA CGA GAG AAG GCT GAG TGG CTG TTG AGG CAG AAA GAG 2052 Ser Thr Pro Lys Arg Glu Lys Ala Glu Trp Leu Leu Arg Gln Lys Glu 605 610 615 620 CAG TTG CAA CAG TGC CAG GCA GAG GAG GAG GCA GGG CTA CTG CGG AGG 2100 Gln Leu Gln Gln Cys Gln Ala Glu Glu Glu Ala Gly Leu Leu Arg Arg 625 630 635 CAA CGC CAG TAC TTT GAG CTT CAG TGT CGC CAA TAC AAG CGC AAG ATG 2148 Gln Arg Gln Tyr Phe Glu Leu Gln Cys Arg Gln Tyr Lys Arg Lys Met 640 645 650 CTA CTG GCT CGG CAC AGC CTA GAC CAG GAC CTG CTT CGA GAG GAC TTG 2196 Leu Leu Ala Arg His Ser Leu Asp Gln Asp Leu Leu Arg Glu Asp Leu 655 660 665 AAT AAG AAA CAG ACA CAG AAG GAC TTG GAG TGT GCT CTG CTG TTA CGG 2244 Asn Lys Lys Gln Thr Gln Lys Asp Leu Glu Cys Ala Leu Leu Leu Arg 670 675 680 CAG CAT GAG GCT ACC CGA GAG CTG GAG CTA CGA CAG CTC CAG GCT GTC 2292 Gln His Glu Ala Thr Arg Glu Leu Glu Leu Arg Gln Leu Gln Ala Val 685 690 695 700 CAG CGC ACA CGT GCT GAA CTC ACC CGC CTT CAG CAC CAG ACA GAG CTA 2340 Gln Arg Thr Arg Ala Glu Leu Thr Arg Leu Gln His Gln Thr Glu Leu 705 710 715 GGC AAC CAG TTG GAG TAC AAC AAG CGA CGG GAG CAA GAG TTG CGG CAG 2388 Gly Asn Gln Leu Glu Tyr Asn Lys Arg Arg Glu Gln Glu Leu Arg Gln 720 725 730 AAG CAC GCG GCC CAG GTT CGC CAG CAG CCC AAG AGC CTC AAA GTA CGT 2436 Lys His Ala Ala Gln Val Arg Gln Gln Pro Lys Ser Leu Lys Val Arg 735 740 745 GCA GGC CAG CTA CCC ATG GGC CTC CCT GCT ACC GGG GCT CTG GGA CCA 2484 Ala Gly Gln Leu Pro Met Gly Leu Pro Ala Thr Gly Ala Leu Gly Pro 750 755 760 CTC AGC ACA GGC ACC CTT AGT GAA GAG CAG CCC TGC TCA TCT GGC CAG 2532 Leu Ser Thr Gly Thr Leu Ser Glu Glu Gln Pro Cys Ser Ser Gly Gln 765 770 775 780 GAG GCA ATC CTG GGC CAA AGG ATG CTG GGA GAG GAG GAG GAA GCA GTG 2580 Glu Ala Ile Leu Gly Gln Arg Met Leu Gly Glu Glu Glu Glu Ala Val 785 790 795 CCA GAG AGA ATG ATT CTG GGA AAG GAA GGG ACT ACT TTG GAG CCA GAG 2628 Pro Glu Arg Met Ile Leu Gly Lys Glu Gly Thr Thr Leu Glu Pro Glu 800 805 810 GAG CAG AGG ATT CTG GGG GAA GAA ATG GGA ACC TTT AGT TCC AGC CCA 2676 Glu Gln Arg Ile Leu Gly Glu Glu Met Gly Thr Phe Ser Ser Ser Pro 815 820 825 CAA AAA CAT AGG AGT CTG GTT AAT GAG GAA GAT TGG GAT ATA TCT AAA 2724 Gln Lys His Arg Ser Leu Val Asn Glu Glu Asp Trp Asp Ile Ser Lys 830 835 840 GAA ATG AAG GAG AGT AGA GTC CCA TCC CTG GCA TCC CAG GAG AGA AAT 2772 Glu Met Lys Glu Ser Arg Val Pro Ser Leu Ala Ser Gln Glu Arg Asn 845 850 855 860 ATT ATT GGC CAG GAA GAG GCT GGG GCA TGG AAT CTG TGG GAG AAG GAG 2820 Ile Ile Gly Gln Glu Glu Ala Gly Ala Trp Asn Leu Trp Glu Lys Glu 865 870 875 CAT GGA AAC CTT GTG GAT ATG GAG TTC AAG CTT GGC TGG GTC CAG GGT 2868 His Gly Asn Leu Val Asp Met Glu Phe Lys Leu Gly Trp Val Gln Gly 880 885 890 CCA GTT CTG ACT CCA GTG CCT GAG GAG GAA GAG GAG GAG GAA GAG GAG 2916 Pro Val Leu Thr Pro Val Pro Glu Glu Glu Glu Glu Glu Glu Glu Glu 895 900 905 GGA GGG GCT CCA ATT GGA ACC CCC AGG GAC CCT GGA GAT GGC TGT CCT 2964 Gly Gly Ala Pro Ile Gly Thr Pro Arg Asp Pro Gly Asp Gly Cys Pro 910 915 920 TCC CCA GAT ATC CCC CCA GAG CCA CCT CCA TCA CAT CTG AGA CAG TAC 3012 Ser Pro Asp Ile Pro Pro Glu Pro Pro Pro Ser His Leu Arg Gln Tyr 925 930 935 940 CCT GCT AGC CAG CTT CCT GGA TTC TTG TCT CAT GGC CTC CTG ACT GGC 3060 Pro Ala Ser Gln Leu Pro Gly Phe Leu Ser His Gly Leu Leu Thr Gly 945 950 955 CTC TCC TTT GCA GTG GGG TCC TCC TCT GGC CTC TTG CCC CTA CTA CTT 3108 Leu Ser Phe Ala Val Gly Ser Ser Ser Gly Leu Leu Pro Leu Leu Leu 960 965 970 CTG CTG CTA CTC CCA TTG CTG GCA CCC AGG TGG AGG TGG CTT GCA GGC 3156 Leu Leu Leu Leu Pro Leu Leu Ala Pro Arg Trp Arg Trp Leu Ala Gly 975 980 985 AGC ACT GCT GGC CCT T GAGGTAGGAC TAGTGGGCCT GGGGGCTTCA 3202 Ser Thr Ala Gly Pro 990 TACCTGTTCC TTTGTACAGC TCTACACCTG CCACCCAGTC TGTTCTTACT CCTGGCTCAG 3262 GGCACTGCAC TGGGGGCTGT CCTTAGCCTG AGCTGGCGCA GAGGCCTTAT GGGTGTGCCT 3322 CTGGGCCTTG GGGCTGCCTG GCTCCTAGCT TGGCCCAGCC TGGCTTTACC TCTGGCAGCT 3382 ATGGCGGCTG GGGGCAAATG GGTACGGCAG CAAGGCCCCC AGATGCGTCG GGGCATCTCT 3442 CGACTCTGGT TGCGGGTTCT GCTACGCCTG TCACCCATGG TCTTTCGGGC CCTACAGGGC 3502 TGTGCGGCTG TGGGAGACCG GGGGCTGTTT GCCCTGTACC CTAAGACCAA TAAGAATGGT 3562 TTCCGAAGTC GACTGCCTGT CCCTTGGCCC CGTCAGGGAA ATCCTCGCAC TACACAGCAC 3622 CCACTAGCTC TGTTAGCAAG AGTTTGGGCT CTGTGCAAGG GCTGGAACTG GCGCCTAGCA 3682 CGGGCTAGCC ATAGATTAGC TTCTTGTTTG CCCCCCTGGG CTGTTCATAT ACTAGCTAGC 3742 TGGGGCCTGC TTAAGGGTGA AAGGCCCAGT CGGATCCCTC GGCTGCTACC GCGAAGCCAA 3802 CGCCGTCTTG GGCTCTCAGC TTCCCGACAG CTACCACCAG GGACTGTAGC TGGGCGGAGA 3862 TCTCAGACCC GCAGGGCCCT GCCTCCCTGG AGGTAACCAG TTCTAACCCT CCACCCAAAT 3922 TTAGGGCATT GAGCACTTTA TCTCCCATGA CTCAGTAAAG TCTCTCCAGT CCCTTGGCCT 3982 CTCCTCCCCT TCTGACCTTT CTTCCTCAGT ATGTTTCCCC AGGTCCAATC CCAGCCCCAG 4042 ATGTAGATTT CTAGACAGGC AGCCTCCTCT ACTGTGGAGT CCAGAATGAC ACTCTTGTGT 4102 TTTCCCCAGT CCCCTAAGTT ATTGCTGTCC CCTGCTGTGT GTGTGCTCAT CCTCACCCTC 4162 ATCGGCTCAG GCCTGGGGCC AGGGGTGGCA GGGAGGGAAG TCATGGGGGT TTTCCCTCTT 4222 TGATTTTGTT TTTCTGTCTC CCTTCCAACC TGTCCCCTTC CCCTCCACCA AAAGAGAAAA 4282 AAAAAAAAAA AAAA 4296 993 amino acids amino acid linear protein 4 Met Pro Ala Gly Gly Arg Ala Gly Ser Leu Lys Asp Pro Asp Val Ala 1 5 10 15 Glu Leu Phe Phe Lys Asp Asp Pro Glu Lys Leu Phe Ser Asp Leu Arg 20 25 30 Glu Ile Gly His Gly Ser Phe Gly Ala Val Tyr Phe Ala Arg Asp Val 35 40 45 Arg Asn Ser Glu Val Val Ala Ile Lys Lys Met Ser Tyr Ser Gly Lys 50 55 60 Gln Ser Asn Glu Lys Trp Gln Asp Ile Ile Lys Glu Val Arg Phe Leu 65 70 75 80 Gln Lys Leu Arg His Pro Asn Thr Ile Gln Tyr Arg Gly Cys Tyr Leu 85 90 95 Arg Glu His Thr Ala Trp Leu Val Met Glu Tyr Cys Leu Gly Ser Ala 100 105 110 Ser Asp Leu Leu Glu Val His Lys Lys Pro Leu Gln Glu Val Glu Ile 115 120 125 Ala Ala Val Thr His Gly Ala Leu Gln Gly Leu Ala Tyr Leu His Ser 130 135 140 His Asn Met Ile His Arg Asp Val Lys Ala Gly Asn Ile Leu Leu Ser 145 150 155 160 Glu Pro Gly Leu Val Lys Leu Gly Asp Phe Gly Ser Ala Ser Ile Met 165 170 175 Ala Pro Ala Asn Ser Phe Val Gly Thr Pro Tyr Trp Met Ala Pro Glu 180 185 190 Val Ile Leu Ala Met Asp Glu Gly Gln Tyr Asp Gly Lys Val Asp Val 195 200 205 Trp Ser Leu Gly Ile Thr Cys Ile Glu Leu Ala Glu Arg Lys Pro Pro 210 215 220 Leu Phe Asn Met Asn Ala Met Ser Ala Leu Tyr His Ile Ala Gln Asn 225 230 235 240 Glu Ser Pro Ala Leu Gln Ser Gly His Trp Ser Glu Tyr Phe Arg Asn 245 250 255 Phe Val Asp Ser Cys Leu Gln Lys Ile Pro Gln Asp Arg Pro Thr Ser 260 265 270 Glu Val Leu Leu Lys His Arg Phe Val Leu Arg Glu Arg Pro Pro Thr 275 280 285 Val Ile Met Asp Leu Ile Gln Arg Thr Lys Asp Ala Val Arg Glu Leu 290 295 300 Asp Asn Leu Gln Tyr Arg Lys Met Lys Lys Ile Leu Phe Gln Glu Ala 305 310 315 320 Pro Asn Gly Pro Gly Ala Glu Ala Pro Glu Glu Glu Glu Glu Ala Glu 325 330 335 Pro Tyr Met His Arg Ala Gly Thr Leu Thr Ser Leu Glu Ser Ser His 340 345 350 Ser Val Pro Ser Met Ser Ile Ser Ala Ser Ser Gln Ser Ser Ser Val 355 360 365 Asn Ser Leu Ala Asp Ala Ser Asp Asn Glu Glu Glu Glu Glu Glu Glu 370 375 380 Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Gly Pro Glu Ser Arg Glu 385 390 395 400 Met Ala Met Met Gln Glu Gly Glu His Thr Val Thr Ser His Ser Ser 405 410 415 Ile Ile His Arg Leu Pro Gly Ser Asp Asn Leu Tyr Asp Asp Pro Tyr 420 425 430 Gln Pro Glu Met Thr Pro Gly Pro Leu Gln Pro Pro Ala Ala Pro Pro 435 440 445 Thr Ser Thr Ser Ser Ser Ser Ala Arg Arg Arg Ala Tyr Cys Arg Asn 450 455 460 Arg Asp His Phe Ala Thr Ile Arg Thr Ala Ser Leu Val Ser Arg Gln 465 470 475 480 Ile Gln Glu His Glu Gln Asp Ser Ala Leu Arg Glu Gln Leu Ser Gly 485 490 495 Tyr Lys Arg Met Arg Arg Gln His Gln Lys Gln Leu Leu Ala Leu Glu 500 505 510 Ser Arg Leu Arg Gly Glu Arg Glu Glu His Ser Gly Arg Leu Gln Arg 515 520 525 Glu Leu Glu Ala Gln Arg Ala Gly Phe Gly Thr Glu Ala Glu Lys Leu 530 535 540 Ala Arg Arg His Gln Ala Ile Gly Glu Lys Glu Ala Arg Ala Ala Gln 545 550 555 560 Ala Glu Glu Arg Lys Phe Gln Gln His Ile Leu Gly Gln Gln Lys Lys 565 570 575 Glu Leu Ala Ala Leu Leu Glu Ala Gln Lys Arg Thr Tyr Lys Leu Arg 580 585 590 Lys Glu Gln Leu Lys Glu Glu Leu Gln Glu Asn Pro Ser Thr Pro Lys 595 600 605 Arg Glu Lys Ala Glu Trp Leu Leu Arg Gln Lys Glu Gln Leu Gln Gln 610 615 620 Cys Gln Ala Glu Glu Glu Ala Gly Leu Leu Arg Arg Gln Arg Gln Tyr 625 630 635 640 Phe Glu Leu Gln Cys Arg Gln Tyr Lys Arg Lys Met Leu Leu Ala Arg 645 650 655 His Ser Leu Asp Gln Asp Leu Leu Arg Glu Asp Leu Asn Lys Lys Gln 660 665 670 Thr Gln Lys Asp Leu Glu Cys Ala Leu Leu Leu Arg Gln His Glu Ala 675 680 685 Thr Arg Glu Leu Glu Leu Arg Gln Leu Gln Ala Val Gln Arg Thr Arg 690 695 700 Ala Glu Leu Thr Arg Leu Gln His Gln Thr Glu Leu Gly Asn Gln Leu 705 710 715 720 Glu Tyr Asn Lys Arg Arg Glu Gln Glu Leu Arg Gln Lys His Ala Ala 725 730 735 Gln Val Arg Gln Gln Pro Lys Ser Leu Lys Val Arg Ala Gly Gln Leu 740 745 750 Pro Met Gly Leu Pro Ala Thr Gly Ala Leu Gly Pro Leu Ser Thr Gly 755 760 765 Thr Leu Ser Glu Glu Gln Pro Cys Ser Ser Gly Gln Glu Ala Ile Leu 770 775 780 Gly Gln Arg Met Leu Gly Glu Glu Glu Glu Ala Val Pro Glu Arg Met 785 790 795 800 Ile Leu Gly Lys Glu Gly Thr Thr Leu Glu Pro Glu Glu Gln Arg Ile 805 810 815 Leu Gly Glu Glu Met Gly Thr Phe Ser Ser Ser Pro Gln Lys His Arg 820 825 830 Ser Leu Val Asn Glu Glu Asp Trp Asp Ile Ser Lys Glu Met Lys Glu 835 840 845 Ser Arg Val Pro Ser Leu Ala Ser Gln Glu Arg Asn Ile Ile Gly Gln 850 855 860 Glu Glu Ala Gly Ala Trp Asn Leu Trp Glu Lys Glu His Gly Asn Leu 865 870 875 880 Val Asp Met Glu Phe Lys Leu Gly Trp Val Gln Gly Pro Val Leu Thr 885 890 895 Pro Val Pro Glu Glu Glu Glu Glu Glu Glu Glu Glu Gly Gly Ala Pro 900 905 910 Ile Gly Thr Pro Arg Asp Pro Gly Asp Gly Cys Pro Ser Pro Asp Ile 915 920 925 Pro Pro Glu Pro Pro Pro Ser His Leu Arg Gln Tyr Pro Ala Ser Gln 930 935 940 Leu Pro Gly Phe Leu Ser His Gly Leu Leu Thr Gly Leu Ser Phe Ala 945 950 955 960 Val Gly Ser Ser Ser Gly Leu Leu Pro Leu Leu Leu Leu Leu Leu Leu 965 970 975 Pro Leu Leu Ala Pro Arg Trp Arg Trp Leu Ala Gly Ser Thr Ala Gly 980 985 990 Pro 414 base pairs nucleic acid single linear 5 ACGANTCACC AGTTGGAAGT TACTCCAAAG AATGAGCACA AAACAATCTT AAAGACACTG 60 AAAGATGAGC AGACAAGAAA ACTTGCCATT TNGGCAGAGC AGTATGAACA GAGTATAAAT 120 GAAATGATGG CCTCTCANGC GTTACGGCTA GATGAGGCTC AAGAAGCAGA ATGCCAGGCC 180 TTGAGGCTAC AGCTCCAGCA GGAAATGGAG CTGCTCAACG CCTACCAGAG CAAAATCAAG 240 ATGCAAACAG AGGCACAACA TGAACGTGAG CTCCAGAAGC TAGAGCAGAG AGTGTCTCTG 300 CGCAGAGCAC ACCTTGAGCA GAAGATTGAA GAGGAGCTGG CTGCCCTTCA GAAGGAACGC 360 AGCGAGAGAA TAAAGAACCT ATTGGAAAGG CAAGAGCGAG AGATTGGAAA CTTT 414 314 base pairs nucleic acid single linear 6 GAACAAAGTC ATGCCTTAAT AGTTCTGCTG ATGTTGGCCT TTCCTGAGGT ATTTTCTGCA 60 AGCAGTAATC AACAAATCTC CTAAAGGAGT CTGTCCATTC ATTAGACTGT AACGTTGGGG 120 AGTCATTCTG GGCAATGTGA TATAAGGCAC TCATTGCATT CATGTTGAAA AGGGGCGGCT 180 TCCGTTCCGC CAATTCAATA CAAGTGATGC CAAGTGACCA AATATCAACT TTCCCATCAT 240 ACTGTCCTTC ATCCATAGCT AAGATCACCT CTGGAGCCAT CCAGTAAGGT GTGCCCACGA 300 AGGAGTTGGC CAGG 314 370 base pairs nucleic acid single linear 7 ACCAAATTCC CAAATCCCAT TCTGAGGCTC TCCATGTCAA AAGTTTCAAT CTCTCGCTCT 60 TGCCTTTCCA ATAGGTTCTT TATTCTCTCG CTGCGTTCCT TCTGAAGGGC AGCCAGCTCC 120 TCTTCAATCT TCTGCTCAAG GTGTGGTCTG CGCAGAGACA CTCTCTGCTC TAGCTTCTGG 180 AGCTCACGTT CATGTTGTGC CTCTGTTNGN ATCTTGATTT GGNTCTGGTA GGCGTTGAGC 240 AGCTCCATTT CCTGCTGGAG CTGTAGCCTC AAGGCCTGGC ATTCTGCTTC TTGAGCCTCA 300 TCTAGCCGTA ACGCTTGAGA GGCCATCATT TCATTTATAC TCTGTTCATA CTGCTCTGCC 360 AAAATGGCAA 370 190 base pairs nucleic acid single linear 8 CAACAGCAGA AAAACTTAAA GGCCATGGAA ATGCAAATTA AAAAACAGTT TCAGGACACT60 TGCAAAGTAC AGACCAAACA GTATAAAGCA CTCAAGAATC ACCAGTTGGA AGTTACTCC120 AAGAATGAGC ACAAAACAAT CTTAAAGACA CTGAAAGATG AGCAGACAAG AAAACTTGC180 ATTTTGGCAG 190 65 base pairs nucleic acid single linear 9 GAGCAGTATG AACAGAGTAT AAATGAAATG ATGGCCTCTC AAGCGTTACG GCTAGATGAG 60 GCTCA 65 219 base pairs nucleic acid single linear 10 ACGAGTCCCC CCGAGAGCTA GAGTACAGGC AGCTGCACAC GTTACAGAAG CTACGCATGG 60 ATCTGATCCG TTTACAGCAC CAGACGGAAC TGGAAAACCA GCTGGAGTAC AATAAGAGGC 120 GAGAAAGAGA ACTGCACAGA AAGCATGTCA TGGAACTTCG GCAACAGCCA AAAAACTTAA 180 AGGCCATGGA ANTGCAATTT AAAAAACAGT TCCAGGAAA 219 85 base pairs nucleic acid single linear 11 GTGCATATGG TATATTTNAT TCATTTTTGT AAAGCGTTCT GTTTTGTGTT TACTAATTGG 60 GATGTCATAG TACTTGGCTG CCGGG 85 46 base pairs nucleic acid single linear 12 CTCACTTGGG TACTACAGTG TGGAAGCTGA GTGCATATGG TATATT 46 116 base pairs nucleic acid single linear 13 GATATTTGGT CATTGGGTAT CACGTGTATA GAGCTGGCCG AACGTCGTCC ACCATTGTTC 60 AGTATGAATG CAATGTCTGC CCTCTACCAT ATTGCTCAAA ATGATCCTCC AACTCT 116 118 base pairs nucleic acid single linear 14 CTGAAAGGCC TGGATTATCT GCACTCAGAG CGCAAGATCC ACCGAGATAT CAAAGCTGCC 60 AACGTGCTGC TCTCGGAGCA GGGTGATGTG AAGATGGCAG ACTTCGGTGT GGCTGGCA 118 110 base pairs nucleic acid single linear 15 GACCCAGAGG AACTCTTCAC CAAGCTTGAC CGCATTGGCA AAGGCTCATT TGGGGAGGTG 60 TACAAGGGGA TCGACAACCA CACCAAGGAA GTGGTGGCCA TCAAGATCAT 110 134 base pairs nucleic acid single linear 16 TCAGGATTCT GGAGCTCTGG AGTTCCATTA GTGGCTATCA GATACAATGC CCTGAGTGGA 60 TTTTCATTAA GGTAAGGGGG TTCACCTTCC ACCATTTCAA TTGCCATAAT TCCAAGAGAC 120 CAGATATCAA CTTT 134 278 amino acids amino acid <Unknown> linear 17 Met Ala Pro Ala Val Leu Gln Lys Pro Gly Val Ile Lys Asp Pro Se 1 5 10 15 Ile Ala Ala Leu Phe Ser Asn Lys Asp Pro Glu Gln Asp Leu Arg Gl 20 25 30 Ile Gly His Gly Ser Phe Gly Ala Val Tyr Phe Ala Tyr Asp Lys Ly 35 40 45 Asn Glu Gln Thr Val Ala Ile Lys Lys Met Asn Phe Ser Gly Lys Gl 50 55 60 Ala Val Glu Lys Trp Asn Asp Ile Leu Lys Glu Val Ser Phe Leu As 65 70 75 80 Thr Val Val His Pro His Ile Val Asp Tyr Lys Ala Cys Phe Leu Ly 85 90 95 Asp Thr Thr Cys Trp Leu Val Met Glu Tyr Cys Ile Gly Ser Ala Al 100 105 110 Asp Ile Val Asp Val Leu Arg Lys Gly Met Arg Glu Val Glu Ile Al 115 120 125 Ala Ile Cys Ser Gln Thr Leu Asp Ala Leu Arg Tyr Leu His Ser Le 130 135 140 Lys Arg Ile His Arg Asp Ile Lys Ala Gly Asn Ile Leu Leu Ser As 145 150 155 160 His Ala Ile Val Lys Leu Ala Asp Phe Gly Ser Ala Ser Leu Val As 165 170 175 Pro Ala Gln Thr Phe Ile Gly Thr Pro Phe Phe Met Ala Pro Glu Va 180 185 190 Ile Leu Ala Met Asp Glu Gly His Tyr Thr Asp Arg Ala Asp Ile Tr 195 200 205 Ser Leu Gly Ile Thr Cys Ile Glu Leu Ala Glu Arg Arg Pro Pro Le 210 215 220 Phe Ser Met Asn Ala Met Ser Ala Leu Tyr His Ile Ala Gln Asn As 225 230 235 240 Pro Pro Thr Leu Ser Pro Ile Asp Thr Ser Glu Gln Pro Glu Trp Se 245 250 255 Leu Glu Phe Val Gln Phe Ile Asp Lys Cys Leu Arg Lys Pro Ala Gl 260 265 270 Glu Arg Met Ser Ala Glu 275 273 amino acids amino acid <Unknown> linear 18 Arg Glu Glu Arg Glu Arg Arg Lys Lys Gln Leu Tyr Ala Lys Leu As 1 5 10 15 Glu Ile Cys Ser Asp Gly Asp Pro Ser Thr Lys Tyr Ala Asn Leu Va 20 25 30 Lys Ile Gly Gln Gly Ala Ser Gly Gly Val Tyr Thr Ala Tyr Glu Il 35 40 45 Gly Thr Asn Val Ser Val Ala Ile Lys Gln Met Asn Leu Glu Lys Gl 50 55 60 Pro Lys Lys Glu Leu Ile Ile Asn Glu Ile Leu Val Met Lys Gly Se 65 70 75 80 Lys His Pro Asn Ile Val Asn Phe Ile Asp Ser Tyr Val Leu Lys Gl 85 90 95 Asp Leu Trp Val Ile Met Glu Tyr Met Glu Gly Gly Ser Leu Thr Va 100 105 110 Asp Val Val Thr His Cys Ile Leu Thr Glu Gly Gln Ile Gly Ala Va 115 120 125 Cys Arg Glu Thr Leu Ser Gly Leu Glu Phe Leu His Ser Lys Gly Va 130 135 140 Leu His Arg Asp Ile Lys Ser Asp Asn Ile Leu Leu Ser Met Glu Gl 145 150 155 160 Asp Ile Lys Leu Thr Asp Phe Gly Phe Cys Ala Gln Ile Asn Glu Le 165 170 175 Asn Leu Lys Arg Thr Thr Met Val Gly Thr Pro Tyr Trp Met Ala Pr 180 185 190 Glu Val Val Ser Arg Lys Glu Tyr Gly Pro Lys Val Asp Ile Trp Se 195 200 205 Leu Gly Ile Met Ile Ile Glu Met Ile Glu Gly Glu Pro Pro Tyr Le 210 215 220 Asn Glu Thr Pro Leu Arg Ala Leu Tyr Leu Ile Ala Thr Asn Gly Th 225 230 235 240 Pro Lys Leu Lys Glu Pro Glu Asn Leu Ser Ser Ser Leu Lys Lys Ph 245 250 255 Leu Asp Trp Cys Leu Cys Cys Val Glu Pro Glu Asp Arg Ala Ser Al 260 265 270 Thr 33 base pairs nucleic acid single linear - 24 /note= “Where N is inosine” - 31 /note= “Where N is inosine” 19 GACGCTGGAT CCAAAGATAC TGGNCAAGGG NGC 33 21 base pairs nucleic acid single linear - /note= “Where N is inosine” - /note= “Where N is inosine” - 13 /note= “Where N is inosine” - 16 /note= “Where N is inosine” - 19 /note= “Where N is inosine” 20 GGNGTNCCAG TTNGTNGCNA T 21 28 base pairs nucleic acid single linear - 11 /note= “Where N is inosine” - 14 /note= “Where N is inosine” - 18 /note= “Where N is inosine” 21 AAAGGAAGCA NAGNCAGNAA CGGAAGAT 28 30 base pairs nucleic acid single linear - 19 /note= “Where N is inosine” - 22 /note= “Where N is inosine” 22 GACGCTGAAT TCACCTTCNG GNGCCATCCA 30 20 amino acids amino acid <Unknown> linear 23 Thr Lys Asp Ala Val Arg Glu Leu Asp Asn Leu Gln Tyr Arg Lys Me 1 5 10 15 Lys Lys Leu Leu 20 19 amino acids amino acid <Unknown> linear 24 Lys Lys Glu Leu Asn Ser Phe Leu Glu Ser Gln Lys Arg Glu Tyr Ly 1 5 10 15 Leu Arg Lys 20 amino acids amino acid <Unknown> linear 25 Arg Glu Leu Arg Glu Leu Glu Gln Arg Val Ser Leu Arg Arg Ala Le 1 5 10 15 Leu Glu Gln Lys 20 8 amino acids amino acid <Unknown> linear 26 His Arg Asp Ile Lys Ala Gly Asn 1 5 

What is claimed is:
 1. An isolated polynucleotide comprising a nucleotide sequence provided in or complimentary to SEQ ID NO:1 or SEQ ID NO:3.
 2. An isolated polynucleotide comprising a nucleotide sequence that encodes the polypeptide provided in SEQ ID NO:2, SEQ ID NO:4, or a variant thereof that is at least 90% identical to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, wherein the polynucleotide encodes a polypeptide capable of phosphorylating MEK3.
 3. An isolated polynucleotide comprising a nucleotide sequence that encodes amino acids 1-416 of SEQ ID NO:2 or SEQ ID NO:4.
 4. An isolated polynucleotide comprising a nucleotide sequence provided in or complimentary to SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16, wherein the polynucleotide encodes a polypeptide capable of phosphorylating MEK3.
 5. An isolated polynucleotide that hybridizes to the polynucleotide of claim 1, 2, 3, or 4 after an incubation at 50° C.-65° C. in 5×SSC followed by two 20 minute washes at 65° C. in each of 2×SSC containing 0.1% SDS, 0.5×SSC containing 0.1% SDS, and 0.2×SSC containing 0.1% SDS, wherein the polynucleotide encodes a polypeptide capable of phosphorylating MEK3.
 6. A polynucleotide according to claim 1, 2, 3, or 4, wherein the polynucleotide encodes a polypeptide that is a constitutively active variant of a TAO polypeptide.
 7. A recombinant expression vector comprising the polynucleotide according to claim 1, 2, 3, or
 4. 8. A host cell containing the expression vector according to claim
 7. 9. A pharmaceutical composition comprising: (a) a polynucleotide according to claim 1, 2, 3, or 4; and (b) a physiologically acceptable carrier.
 10. A polynucleotide according to 5, wherein the polynucleotide encodes a polypeptide that is a constitutively active variant of a TAO polypeptide.
 11. A recombinant expression vector comprising the polynucleotide according to claim
 5. 12. A host cell containing the expression vector according to claim
 11. 13. A pharmaceutical composition comprising: (a) a polynucleotide according to claim 5; and (b) a physiologically acceptable carrier.
 14. An isolated polynucleotide comprising a nucleotide sequence that encodes a polypeptide that is at least 90% identical to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, wherein the polynucleotide encodes a polypeptide that is a constitutively inactive variant of a TAO polypeptide. 